Review of harmful gastrointestinal effects of carrageenan in animal experiments. (Research Review).
During the latter half of the twentieth century, inflammatory bowel disease and gastrointestinal malignancy have been major causes of morbidity and mortality in the United States. Even with improvements in treatment and cancer screening, colorectal cancer remains the second leading cause of cancer mortality in the United States. The Western diet has been considered a possible source of inflammatory bowel disease and colorectal malignancy, and intensive efforts have been undertaken to study the impact of specific constituents of the Western diet, such as fiber and fat (1-3).
One food additive, carrageenan, has been associated with induction and promotion of intestinal neoplasms and ulcerations in numerous animal experiments; however, carrageenan remains a widely used food additive. In 1982, the International Agency for Research on Cancer (IARC) (4) designated degraded carrageenan as Group 2B, noting sufficient evidence for the carcinogenicity of degraded carrageenan in animal models to infer that "in the absence of adequate data on humans, it is reasonable, for practical purposes, to regard chemicals for which there is sufficient evidence of carcinogenicity in animals as if they presented a carcinogenic risk to humans" (p. 90). The National Research Council has noted this designation for degraded carrageenan in their 1996 monograph (5). Recognizing the impact of carrageenan in animal models, several European and British investigators have advised against the continued use of carrageenan in food (6-11). Several reports have called attention to the problems associated with carrageenan consumption (6-11).
Extracted from red seaweed, carrageenan has been used in food products for centuries and was patented as a food additive for use in the United States in the 1930s. It has been used widely as a food additive, contributing to the texture of a variety of processed foods. It has also been used as a laxative, as treatment for peptic ulcer disease, and as a component of pharmaceuticals, toothpaste, aerosol sprays, arm other products (12-15). In 1959, carrageenan was granted GRAS (Generally Regarded as Safe) status in the United States. GRAS substances are permitted to be incorporated into food products as long as good manufacturing processes are used and the substance is used only in sufficient quantity to achieve the desired effect (16,17).
In the United States, the status of carrageenan was reconsidered by the Food and Drug Administration, and an amendment to the Code of Federal Regulations for the food additive carrageenan was proposed in 1972 (18). To diminish the public's exposure to degraded carrageenan, the amendment supported inclusion of an average molecular weight for carrageenan of 100,000 and a minimum viscosity of 5 centipoises (cps) under specified conditions. However, the actual regulation was not amended, although several publications indicated that it had been modified (7,8,19-23). In 1979, the proposal to include the average molecular weight requirement of 100,000 and the associated viscosity requirement in the Code of Federal Regulations was withdrawn. It was anticipated that a new rule-making proposal on carrageenan that would comprehensively address all food safety aspects of carrageenan and its salts would be published in about a year, but this has not been forthcoming (24,25). The proposal withdrawal referred to interim specifications for food-grade carrageenan using the Food Chemical Codex; these include a viscosity stipulation, but no average molecular weight requirement (26).
In the Food Chemicals Codex and supplements, carrageenan is described with attention to specific requirements for its identification and tests of its properties, including its sulfate content, heavy metal content, solubility in water, content of acid-insoluble matter, and viscosity [a 1.5% solution is to have viscosity [is greater than or equal to] 5 cps at 75 [degrees] C] (26,27). Although the viscosity is stipulated, viscosity may not adequately protect food-grade carrageenan from contamination by the lower molecular weight degraded carrageenans that IARC has denoted as Group 2B. Because undegraded carrageenan may have molecular weight in the millions, the actual viscosities of commercial carrageenans range from about 5 to 800 cps when measured at 1.5% at 75 [degrees] C (14). Native carrageenan has molecular weights of 1.5 x [10.sup.6]-2 x [10.sup.7] (28); poligeenan or degraded carrageenan is described as having average molecular weight of 20,000-30,000 (4). The average molecular weight of poligeenan has been described elsewhere as 10,000-20,000, but extending up to 80,000 (29). Food-grade carrageenan has been described as having average molecular weight of 200,000-400,000 (29), and elsewhere as having molecular weight of 100,000-800,000 (19). Furcelleran (or furcellaran), a degraded carrageenan of molecular weight 20,000-80,000, has a sulfate content of 8-19% (12,17). No viscosity or minimum average molecular weight was designated for furcelleran in the 1972 or 1979 Federal Register documents (18,24). In the Food Chemical Codex (fourth edition), a 1.5% solution of furcelleran at 75 [degrees] C is described as having minimum viscosity of 5 cps (27).
Today, carrageenan is still included among the food additives designated GRAS in the Code of Federal Regulations. The stipulations for its use include the following: a) it is a sulfated polysaccharide, the dominant hexose units of which are galactose and anhydrogalactose; b) range of sulfate content is 20-40% on a dry-weight basis; c) the food additive is used or intended for use in the amount necessary for an emulsifier, stabilizer, or thickener in foods, except for those standardized foods that do not provide for such use; d) to assure safe use of the additive, the label and labeling of the additive shall bear the name of the additive, carrageenan. Also included are similar standards for carrageenan salts and for furcelleran and furcelleran salts (30). In 1999-2000, approved uses for carrageenan were extended to include additional incorporation into food and medicinal products, including both degraded and undegraded carrageenan in laxatives (31-33).
For use in experimental models, degraded carrageenan (poligeenan) is derived from carrageenan by acid hydrolysis, frequently by a method developed by Watt et al. (34). This method is expected to yield a degraded carrageenan of average molecular weight 20,000-30,000 (35). Experiments demonstrate that reaction conditions similar to those of normal digestion can lead to the formation of degraded carrageenan (9-11). In addition, experimental data have revealed the contamination of food-grade carrageenan by substantial amounts of degraded carrageenan (10). Also, some bacteria are known to hydrolyze carrageenan and form low molecular weight derivatives (36-40).
The sections that follow and the accompanying tables summarize many experimental observations with regard to the intestinal effects of carrageenan. In addition, I review possible mechanisms for production of degraded carrageenan from undegraded carrageenan under physiologic conditions, as well as evidence that provides a basis for the mechanism of carrageenan's effects and for the reconsideration of the safety of carrageenan in the human diet.
Characteristics of Carrageenan
Three forms of carrageenan predominate, known as kappa, iota, and lambda. All have similar D-galactose backbones (alternating [alpha]-1,3 to [beta]-1,4 linkages), but they differ in degree of sulfation, extent of branching, solubility, cation binding, and ability to form gels under different conditions. [lambda]-Carrageenan is the least branched and the least gel forming; it is readily soluble at cold temperatures, in contrast to [kappa]- or [iota]-carrageenan. Table 1 presents some of the basic characteristics of [kappa], [iota], and [lambda] carrageenan (4,12-15,20-22,31-33,41-44)
In addition to food additive uses, carrageenan has been used in cosmetics, pesticides, and pharmaceuticals, as well as in toothpaste and room deodorizers. It has been used as a treatment of ulcers and as an emulsifier in mineral oil laxatives, liquid petrolatum, and cod liver oil. However, its predominant role has been in food preparations, in which it is used across a wide variety of food groups because of its ability to substitute for fat and its ability to combine easily with milk proteins to increase solubility and improve texture. Hence, it is used in low-calorie formulations of dietetic beverages, infant formula, processed low-fat meats, whipped cream, cottage cheese, ice cream, and yogurt, as well as in other products. From its original use several centuries ago as a thickener in Irish pudding and its incorporation into blancmange, the food additive use has extended widely and cuts across both low-fat and high-fat diets. It is often combined with other gums, such as locust bean gum, to improve the texture of foods (12-14,22,41,42).
In 1977, data obtained by the survey of industry on the use of food additives produced an estimate of daily carrageenan intake of 100 mg for individuals older than 2 years. The 1971 survey of industry had indicated that formula-fed infants in the first 5 months of life had an intake of 108 mg/day (21,43). Informatics, Inc., in a report prepared for the Food and Drug Administration, cited daily carrageenan consumption of 45 mg (19); this is similar to the reported intake of 50 mg/day of carrageenan in France (45). Nicklin and Miller (20) reported intake of 0-1.5 g/day, depending on choice of diet and total food consumed. Although the Food and Nutrition Board of the National Research Council of the National Academy of Sciences of the United States in 1971 initially estimated 367 mg/day for carrageenan intake for individuals older than 2 years in the United States, this was subsequently revised to 11 mg/day. The wide range of estimates may be attributed to inconsistencies in how industry has reported carrageenan production and consumption data, variation in processed food formulations with regard to extent of incorporation of carrageenan, and changes in use of carrageenan in nonfood products. Daily individual consumption of between 50 mg/day and 100 mg/day is consistent with total consumption in the United States of 7,700 metric tons, as estimated for 1997 (46).
The content of carrageenan in several commonly consumed food products is summarized in Table 2. Because manufacturing practices vary and change over time and the food formulae are proprietary, carrageenan content is indicated by a range (12,13,47-49). The content is expressed as the percent by weight of carrageenan used in the production of the food.
Experimental Results in Animal Models
Intestinal lesions after exposure to carrageenan in animal models. Table 3 summarizes the laboratory investigations that associate exposure to carrageenan with the occurrence of intestinal lesions (50-93). Several animals were tested, including guinea pig, rat, monkey, mouse, rabbit, and ferret. The guinea pig seemed most susceptible to ulceration and the rat most susceptible to malignancy. Many studies used exposure to carrageenan in a drinking fluid, at concentrations generally of 1%. Some were feeding studies, in which carrageenan was added to a solid diet. Some studies used gastric or duodenal intubation to ensure intake at a specified level; however, this method may have affected the way that carrageenan was metabolized by gastric acid (74,82-84,91). Feeding of carrageenan with milk may also have affected study results, because carrageenan binds tightly to milk proteins (caseins), affecting its metabolism (12-15, 22,41,42,47). The degraded carrageenan used in most of the experiments had molecular weight from 20,000 to 40,000. Several major findings in relation to neoplasia and ulceration were observed in these animal studies. All of these studies observed the effects of carrageenan in comparison to appropriate control animals.
In the footnote to Table 3, several subdivisions of the table are indicated with citation of the entries from the table. The subdivisions include: a) studies in which carrageenan alone induces abnormal proliferation or malignancy, b) studies in which carrageenan alone induces intestinal ulcerations, c) studies in which carrageenan appears to be a promoter of malignancy in association with another agent, d) studies using a rat model, e) studies using a guinea pig model, f) studies using degraded carrageenan, g) studies using undegraded carrageenan, h) studies indicating uptake of carrageenan into an extraintestinal site(s), i) studies indicating intestinal breakdown of carrageenan into lower molecular weight forms, and j) studies demonstrating ulcerations in rats using degraded carrageenan. In the table, the classification of the carrageenan used in the experiments as [kappa], [lambda], or [iota] is indicated when this information is clear from the original report.
Neoplasia. Wakabayashi and associates (72) demonstrated the appearance of colonic tumors in 32% of rats fed 10% degraded carrageenan in the diet for less than 24 months. The lesions included squamous cell carcinomas, adenocarcinomas, and adenomas. With exposure to 5% degraded carrageenan in drinking water, there was a 100% incidence of colonic metaplasia after 15 months. Metastatic squamous cell carcinoma was observed in retroperitoneal lymph nodes in this experiment. In addition, macrophages that had metachromatic staining consistent with carrageenan uptake were observed in liver and spleen.
Other studies have demonstrated the development of polypoidal lesions and marked, irreversible squamous metaplasia of the rectal mucosa, the extent of which was associated with duration and concentration of carrageenan exposure (67,70). Oohashi et al. (67) observed a 100% incidence of colorectal squamous metaplasia that progressed even after degraded carrageenan intake was discontinued in rats fed 10% degraded carrageenan for 2, 6, or 9 months and sacrificed at 18 months.
Fabian et al. (84) observed adenomatous and hyperplastic polyps as well as squamous metaplasia of the anorectal region and the distal colon in rats given 5% carrageenan as a drinking fluid. Similarly, Watt and Marcus (90) observed hyperplastic mucosal changes and polypoidal lesions in rabbits given carrageenan as drinking fluid for 6-12 weeks at a concentration of 0.1-5%. Focal and severe dysplasia of the mucosal epithelium was observed in rabbits after 28 months of 1% degraded carrageenan in their drinking fluid (58).
Promotion of neoplasia. Several studies demonstrated an increased occurrence of neoplasia in relation to exposure to undegraded or degraded carrageenan and associated exposure to a known carcinogen. Experimental data with undegraded carrageenan included enhanced incidence of colonic tumors in rats treated with azoxymethane (AOM) and nitrosomethylurea (NMU), when carrageenan was added to the diet. Groups of rats received control diet; control diet with 15% carrageenan; 15% carrageenan plus 10 injections of AOM given weekly; carrageenan plus NMU; NMU alone; and AOM alone. AOM or NMU with carrageenan led to 100% incidence of tumors, versus 57% with AOM alone and 69% with NMU alone (p < 0.01). Controls had 0%, and carrageenan alone led to an incidence of 7%. In addition, when undegraded carrageenan was combined with AOM, there was a 10-fold increase in the number of tumors per rat (73). (Figure 1)
Using undegraded carrageenan as a solid gel at concentration 2.5% for 100 days, Corpet et al. (50) found that after exposure to azoxymethane, there was promotion of aberrant crypt foci by 15% (p = 0.019). Exposure of rats to 6% undegraded carrageenan in the diet for 24 weeks, with 1,2-dimethylhydrazine (1,2-DMH) injections weekly, was associated with an increase in tumors from 40% to 75% and with the more frequent occurrence of larger and proximal tumors (57).
Degraded carrageenan in the diet of rats at a 10% concentration in association with exposure to 1,2-DMH weekly for 15 weeks was associated with an increase in small intestinal tumors from 20% to 50% and in colonic tumors from 45% to 60% (64). Iatropoulos et al. (77) found that in rats given 5% degraded carrageenan in the drinking water for less than 30 weeks in association with injections of 1,2-DMH weekly, there were increases in poorly differentiated adenocarcinomas and in tumors of the ascending and transverse colon, as well as increased proliferation of cells in the deep glandular areas.
Several investigators have measured the effect of carrageenan on thymidine incorporation and colonic cell proliferation. Wilcox et al. (51) observed a 5-fold increase in thymidine kinase activity in colon cells with 5% undegraded or 5% degraded carrageenan. There was an associated 35-fold increase in proliferating cells in the upper third of crypts with degraded carrageenan and an 8-fold increase with undegraded carrageenan (51). With 5% [lambda]-undegraded carrageenan fed to rats for 4 weeks, Calvert and Reicks (55) observed a 4-fold increase in thymidine kinase activity in the distal 12 cm of the colon (p < 0.001). Fath et al. (59) observed a 2-fold increase in colonic epithelial cell proliferation, with increase in labeling indices in both proximal and distal colon and extensive increase of the proliferative compartment in the proximal colon to the upper third of the intestinal crypt, after exposure of mice to 10% degraded carrageenan in drinking water for 10 days.
Ulceration. Many studies have demonstrated significant ulceration of the cecum and/or large intestine after oral exposure to carrageenan in guinea pigs, rabbits, mice, rats, and rhesus monkeys (34,35,53,56,58,59,62,63,65,68,70,71,75, 78-80,82,83,86-93). Ulcerations arose in association with exposure to either degraded or undegraded carrageenan. Lesions occurred initially in the cecum of guinea pigs and rabbits, but could be induced in more distal parts of the colon of the guinea pig, as in an experiment in which carrageenan was introduced directly into the colon after ileotransversostomy (63). In rats, the ulcerative lesions appeared initially in distal colon and rectum (8). Undegraded and degraded carrageenan have been associated with epithelial cell loss and erosions in rats (51,65,70,87,93).
Watt et al. (34) first observed ulcerations in response to carrageenan exposure in animal models more than three decades ago. They noted that 100% of guinea pigs given 2% degraded carrageenan as liquid for 20-30 days had colonic ulcerations and that 75% of the animals > 200 ulcers (34). When guinea pigs were given 1% undegraded carrageenan as liquid for 20-30 days, 80% developed colonic ulcerations (92). The lesions were routinely produced with carrageenan concentrations of 0.1-1%, which is similar to the concentration in a variety of food products (7,12-14).
Grasso et al. (83) demonstrated pinpoint cecal and colonic ulcerations in guinea pigs and rabbits given 5% undegraded, as well as degraded, carrageenan in the diet for 3-5 weeks. Lesions were not observed in ferrets and squirrel monkeys given degraded carrageenan by gastric intubation (83). Other investigators have also observed ulcerations after exposure to either degraded or undegraded carrageenan (75,88). Engster and Abraham (75) observed ulceration of cecum in guinea pigs given ??-carrageenan of molecular weight 21,000-107,000, demonstrating ulcerations were also caused by higher molecular weight carrageenan. Cecal ulcerations were not found with exposures to [Kappa] or [lambda] carrageenan of molecular weight varying from 8,500-314,000.
Investigators have noted that carrageenan-induced ulcerations of the colon are dose dependent and related to duration of exposure (52,53,67,68,70,89,90). Kitsukawa et al. (52) observed small epithelial ulcerations in guinea pigs who received carrageenan in their drinking fluid at two days. Olsen and Paulsen (68) observed cecal lesions after 24 hr and confluent ulcerations after 7 days in guinea pigs that ingested a 5% carrageenan solution. In rats, superficial erosions were observed at the anorectal junction at 24 hr after 10% dietary carrageenan (70); these extended more proximally over time. In 5 days of feeding with a 5% carrageenan solution, Jensen et al. (62) observed as many as 111 ulcerations/[cm.sup.2] over the mucosal surface of the cecum in the guinea pig.
Benitz et al. (82) observed a dose effect when degraded carrageenan was given at concentrations of 0.5-2% in drinking fluid to rhesus monkeys for 7-14 weeks. Watt and Marcus (89) observed that in rabbits given 0.1% degraded carrageenan as drinking fluid, 60% of the animals developed ulcerations, whereas 100% of those given 1% carrageenan had ulcerations when exposed for 6-12 weeks.
Resemblance to ulcerative colitis. Several investigators have noted the resemblance between the ulcerative lesions and accompanying inflammatory changes induced by carrageenan and the clinical spectrum of ulcerative colitis (56,94-99). Since the development of the carrageenan-induced model of ulcerative disease of the colon in 1969, carrageenan exposure has been used to model ulcerative colitis and to test for response to different treatments (52,62,100,101).
Clinical features in the experimental animals exposed to carrageenan have included weight loss, anemia, diarrhea, mucous in stools, and visible or occult blood in stools. The absence of small intestinal lesions and the lack of remission and exacerbation are also characteristic features of the carrageenan model (99,102).
Onderdonk (94) discussed the similarity between the carrageenan model of colitis and ulcerative colitis in humans and considered whether animal models for inflammatory bowel disease were also models for intestinal cancer because of the increased risk of colon cancer in individuals with ulcerative colitis. He reviewed the findings from carrageenan-treated animals, including loss of haustral folds, mucosal granularity, crypt abscesses, lymphocytic infiltration, capillary congestion, pseudopolyps, and strictures. Other observations have demonstrated an apparent sequence from colitis to squamous metaplasia and then to tumors of the colorectum (67,72,102). Atypical epithelial hyperplasia in the vicinity of carrageenan-induced ulcerations resembled findings from human ulcerative colitis that provide a link to intestinal neoplasia (86,98).
Proposed mechanism of development of lesions. A common feature observed in the animal models of ulceration in association with carrageenan exposure is macrophage infiltration (35,56,63,65,68,75,76,78-81, 83,84,88,92,102-104). Fibrillar material and metachromatic staining of the macrophages were observed. Notably, the macrophage lysosomes appeared to take up the fibrillar material and to become distorted and vacuolated. It appeared that colonic ulcerations developed as a result of macrophage lysosomal disruption, with release of intracellular enzymes, subsequent macrophage lysis, and release of intracellular contents that provoked epithelial ulceration (75, 76, 79, 84, 85, 88, 105,106). In the rhesus monkey, Mankes and Abraham (76) observed vacuolated macrophages with fibrillar material when the animals were given undegraded carrageenan of molecular weight 800,000 as a 1% solution in their drinking fluid, demonstrating the occurrence of these changes after exposure to undegraded as well as to degraded carrageenan.
In an effort to clarify further the precise pathogenic changes that occurred, Marcus et al. (35) evaluated pre-ulcerative lesions after exposure of guinea pigs to degraded carrageenan for only 2-3 days. The animals received 3% drinking solution of carrageenan, with an average daily carrageenan intake of 5.8 g/kg. Early focal lesions were observed macroscopically in the cecum in only one animal with this brief exposure. However, in all test animals, a diffuse cellular infiltrate, with macrophages and polymorphonuclear leukocytes, was apparent microscopically. Inflammatory changes in the cecum and ascending colon were present in all animals, and in the distal colon and rectum in three of four animals. Metachromatic staining material was noted in the lamina propria of the colon and surface epithelial cells from cecum to rectum, as well as in colonic macrophages. The surface epithelial cells and the macrophages contained vacuoles filled with the metachromatic material, which was not found in the controls and not seen in more advanced lesions in previous studies. These early lesions suggested that the presence of degraded carrageenan within surface epithelial cells might be associated with the subsequent breakdown of the mucosa and to ulceration by a direct toxic effect on the epithelial cells (35).
Hence, a model of mechanical cellular destruction by disruption of lysosomes from carrageenan exposure arises from review of the experimental studies in animals. The observed changes in the lysosomes resemble the characteristic changes observed in some lysosomal storage diseases, in which there is accumulation of sulfated metabolites that cannot be processed further due to sulfatase enzyme deficiency (107-110). Table 4 presents a proposed mechanism of the effects of carrageenan.
Possible role of intestinal bacteria. The relationship between the intestinal microflora and the biologic activity of carrageenan has been reviewed (111,112). Investigators have examined the impact of antibiotics and alteration of the resident microbial flora on the activity of carrageenan. Grasso et al. (83) studied the impact of neomycin treatment on the development of ulcerations by carrageenan. Pretreatment against coliforms failed to attenuate the course of carrageenan-associated ulcerations (80,83). Pretreatment with metronidazole was effective in preventing carrageenan-induced colitis in another experiment, although there was no benefit in established colitis (71). Aminoglycosides administered after carrageenan exposure were associated with reduced mortality, but not with reduction in the number of colonic ulcerations (94). Hirono et al. (65) found increased ulcerations and squamous metaplasia from the anorectal junction to the distal colon in germ-free rats fed 10% carrageenan for less than 63 days.
Additional considerations about the mechanism of action of carrageenan involved the role of production of hydrogen sulfide gas from metabolism of carrageenan in the digestive tract. Because carrageenan is heavily sulfated (up to 40% by weight), bacterial sulfatases and sulfate reductases can produce hydrogen sulfide gas or H[S.sup.-] from carrageenan. Carrageenan, as well as other sulfated polysaccharides, has been shown to stimulate [H.sub.2]S production from fecal slurries (113). Sulfide has been implicated in the development of ulcerative colitis, perhaps attributable to interference with butyrate oxidation by colonic epithelial cells (114,115). Butyrate has been shown to induce intestinal cellular differentiation, suppress intestinal cell growth, and decrease expression of c-myc, among other functions in colonic epithelial cells (116-118).
No fermentation of carrageenan was reported after testing with 14 strains of intestinal bacteria. The increase in sulfide production observed arising from incubation of [kappa]-carrageenan with colonic bacteria demonstrates that intestinal metabolism of carrageenan does occur. However, data pertaining to breakdown of carrageenan by fecal organisms are limited (112,113).
Extraintestinal manifestations of carrageenan exposure. Trace amounts of undegraded carrageenan have been reported to cross the intestinal barrier, with accumulation of label in intestinal lymph nodes (61,74). Several investigators have noted uptake of carrageenan by intestinal macrophages with subsequent migration of these macrophages to lymph nodes, spleen, and liver (61,67,74,78,82,84,85).
In association with carrageenan-induced intestinal ulcerations, Delahunty et al. (56) observed an increased permeability to large molecules, such as [[sup.3]H]PEG (polyethylene glycol)-900. This finding suggested that the intestinal changes induced by carrageenan may be a factor in subsequent absorption of carrageenan or other large molecules.
Other experimental data. Because it can induce acute inflammation, carrageenan has been widely used in experimental models of inflammation, to assess activity of anti-inflammatory drugs and to study mediators of inflammation (4,61,106,119,120). Injected into an experimental site, such as the plantar surface of a rat's paw, pleural cavity, or subcutaneous air bleb, carrageenan induces an inflammatory response, with edema, migration of inflammatory cells, predominantly polymorphonuclear leukocytes, and possibly granuloma formation (61,120). Undegraded carrageenans in vitro can inhibit binding of basic fibroblast growth factor (bFGF), transforming growth factor [beta]-1, and platelet-derived growth factor but not insulin-like growth factor-1 or transforming growth factor-[alpha] (121).
Macrophage injury and destruction caused by carrageenan may be a factor in the reduced cytotoxic lymphocytic response associated with carrageenan exposure in vivo (122). In addition to depression of cell-mediated immunity, impairment of complement activity and of humoral responses have been reported. Prolongation of graft survival and potentiation of tumor growth have been attributed to the cytopathic effect on macrophages (96,123). Because of its effect on T-cells, carrageenan has been studied for its impact on viral infections with herpes simplex virus types 1 and 2 (124) and HIV-1 (125,126), as well as infections with Chlamydia trachomatis (127).
In experimental systems, undegraded carrageenan has produced destruction of several different cell types in addition to macrophages, including small intestine epithelial cell monolayers (54), androgen-dependent malignant prostatic cells (128), bFGF-dependent endothelial cell line (128), rat mammary adenocarcinoma 13762 MAT cells (129), and human mammary myoepithelial cells (130). Lysosomal inclusions and vacuolation have been observed in macrophages, intestinal epithelial cells, and myoepithelial cells exposed to carrageenan (79,85,131).
Injections of carrageenan were noted to induce sarcomas, as well as mammary tumors in animal models, in an early study (132). In other experiments, mammary and testicular tumors have been observed (69,133). Carrageenan has also been noted to have anticoagulant activity, and large systemic doses have been fatal through nephrotoxicity (4).
Mechanisms for Production of Degraded Carrageenan from Undegraded Carrageenan
Gastrointestinal metabolism of carrageenan to form smaller molecular weight components has been observed by several investigators, who reported that carrageenan of high molecular weight changed during intestinal passage, compatible with hydrolysis yielding lower molecular weight components (9,10,74,75).
Under conditions such as might occur in digestion, 17% of food-grade carrageenan degraded to molecular weight < 20,000 in 1 hr at pH 1.2 at 37 [degrees] C. At pH 1.9 for 2 hr at 37 [degrees] C, 10% of the carrageenan had molecular weight less than 20,000 (9). These data suggest that substantial fractions of lower molecular weight carrageenan are likely to arise during normal digestion.
Table 5 presents data with regard to contamination of food-grade carrageenan by lower molecular weight carrageenan. Twenty-five percent of total carrageenans in eight food-grade carrageenans were found to have molecular weight < 100,000, with 9% having molecular weight < 50,000 (9). In addition, several bacteria have been identified that are able to hydrolyze carrageenan into smaller products, including tetracarrabiose. These bacteria, including Cytophaga species and Pseudomonas carrageenovora, are of marine origin; it is unknown whether the human microbial flora can perform similar hydrolysis reactions (36-40,134).
Extent of Human Exposure to Carrageenan
Indirect evidence relating exposure to carrageenan and the occurrence of ulcerative colitis and intestinal neoplasms consists of the similar geographic distribution between higher consumption of carrageenan and higher incidence of inflammatory bowel disease and colorectal cancer. Ulcerative colitis is more common in North America, the United Kingdom, and Scandinavia; and less common in Central and Southern Europe, Asia, and Africa (135). This incidence distribution is similar to distributions for colorectal malignancy and for carrageenan consumption, providing some ecologic evidence to support a potential etiologic role of carrageenan in human disease (46,136).
The reported T[D.sup.50] (tumorigenic dose 50% = the dose rate, in milligrams per kilogram body weight per day, which will halve the probability of remaining tumorless over the life span of the exposed animal) by the Carcinogenic Potency Database for degraded carrageenan is 2,310 mg/kg body weight/day, based on rodent experiments (137,138). This extrapolates to 138.6 grams for a 60-kg individual. If the total carrageenan intake per person in the United States is about 100 mg a day (43), about 9 mg of carrageenan with molecular weight < 50,000 is likely to be ingested through contamination of food-grade carrageenan by degraded carrageenan, and at least 8 mg with molecular weight < 20,000 is likely to arise during normal digestion (simulated by exposure to pH 1.9 with pepsin for 1 hr at 37 [degrees] C). This suggests an average intake of about 10 mg/day of degraded carrageenan for an individual older than 2 years of age in the United States.
An important issue is whether 10 mg/day degraded carrageenan is safe to ingest. By the Delaney clause, no carcinogen should be permitted in food. The Food Quality Protection Act (FQPA) established a usage level for negligible risk associated with pesticide residue in food at 1 ppm (139,140). Applying this standard to the extrapolated T[D.sub.50] for degraded carrageenan for a 60-kg person, the anticipated average intake of 10 mg/day is 70-fold greater than this standard (138.6 g/[10.sup.6]/day). These calculations do not take into consideration possible exposure to furcellaran (molecular weight 20,000-80,000), or the wide range of possible intakes of carrageenan.
Inflammatory bowel disease and colorectal malignancy represent major sources of morbidity and mortality in the United States. A possible factor in the etiology of these pathologies is exposure to carrageenan.
Several investigators have expressed their concerns about the use of undegraded carrageenan in food products (6-10), yet no legislative protection to restrict incorporation of low molecular weight fractions has been enacted. In fact, there has been no substantive review by the Food and Drug Administration of carrageenan since the studies undertaken more than two decades ago. However, there has been increased evidence regarding the cancer-promoting activity of undegraded carrageenan and further confirmation of the carcinogenic potential of degraded carrageenan.
Evidence for the role in carcinogenesis of carrageenan appears to support a nongenotoxic model based on direct toxic effects, for carrageenan has been nonmutagenic in Salmonella mutagenicity testing and nongenotoxic by DNA repair tests (60,102). A model of cellular destruction--from disruption of lysosomes by accumulation of carrageenan by-products or by interference with normal cellular oxidation-reduction processes from sulfate metabolites--emerges from review of the experimental studies. The impact of sulfatases, of either bacterial or human origin, on the metabolism of carrageenan requires further investigation. By interference with the normal intracellular feedback mechanisms associated with arylsulfatase activity, including steroid sulfatase, the highly sulfated carrageenan may have an impact on the availability of active, unsulfated hormones, such as dehydroepiandrosterone, derived from dehydroepiandrosterone-sulfate, and estrone-1, derived from estrone-1 sulfate.
Genetic characteristics that affect sulfatase and hydrolysis reactions as well as the individual intestinal microflora may influence how carrageenan is metabolized and how its effects are manifested. These factors may determine how carrageenan is metabolized differently by different individuals, but these characteristics may not be accessible to manipulation. A basic factor that can be controlled is the intake of carrageenan, which is amenable to dietary modification or food additive regulation.
Although carrageenan is widely used as a food additive for its texture-enhancing properties, other gums, some of which are used in combination with carrageenan, such as locust bean gum, gum arabic, alginate, guar gum, or xanthan gum, potentially can be used alone or in different combinations as substitutes for carrageenan (41,46). Alternatively, higher fat composition can lead to changes in food properties that may compensate for exclusion of carrageenan. Other hydrocolloids that are used as stabilizers and thickeners have not been associated with harmful gastrointestinal effects, and it is reasonable to expect that they could replace carrageenan in many food products. Although the dietary fibers pectin and psyllium affect intestinal motility, ulcerations or neoplasms have not been induced with either these or the other water-soluble polymers used as food additives. In contrast, other highly sulfated polysaccharides, amylopectin sulfate and dextran sulfate sodium, have induced ulcerations and neoplasia, suggesting that the degree of sulfation and polysaccharide molecular weight may be critical for induction of the observed effects (102).
The major pieces of evidence that support an argument to reconsider the advisability of use of carrageenan as a GRAS food additive are:
* Degraded carrageenan is a known carcinogen in animal models
* Undegraded carrageenan is a known co-carcinogen in animal models of carcinogenesis
* In animal models, both degraded and undegraded carrageenan have been associated with development of intestinal ulcerations that resemble ulcerative colitis
* Hydrolysis such as may occur by exposure to gastric acid in the human stomach can lead to the depolymerization of undegraded carrageenan and the availability of degraded carrageenan
* Food-grade carrageenan may be contaminated with low molecular weight, degraded carrageenan that may arise during food processing
* The use of a viscosity measurement to characterize a carrageenan sample is insufficient because the presence of a small number of large molecules (and undegraded carrageenan may have molecular weight in the millions) may obscure a significant low molecular weight fraction.
The potential role of carrageenan in the development of gastrointestinal malignancy and inflammatory bowel disease requires careful reconsideration of the advisability of its continued use as a food additive.
Table 1. Characteristics of carrageenan (4,12-15,27,28,41-49). Chemical composition Hydrocolloid composed of [alpha]-D-1,3 and [beta]-D-1,4 galactose residues that are sulfated at up to 40% of the total weight. Strong negative charge over normal pH range. Associated with ammonium, calcium, magnesium, potassium, or sodium salts. Solubility [lambda] is readily soluble in cold or hot aqueous solution; [kappa] is soluble in hot solution; treatment of aqueous solution with potassium ion precipitates [kappa]-carrageenan. Gel formation [lambda] does not form gels; [lambda] and [iota] form right-handed helices; potassium chloride promotes gel formation of [kappa]; calcium ion promotes gel formation of [iota]. Metabolism Hydrolysis of glycosidic linkages at lower pH, especially pH [less than or equal to] 3.0; also desulfation by sulfatases. Viscosity Near logarithmic increase in viscosity with increasing concentration. Viscosity of food- grade carrageenan defined as not less than 5 cps at 75 [degrees] C for a 1.5% solution; viscosity ranges from 5 to 800 cps for 1.5% solution at 75 [degrees] C. Source Red algae; predominantly aqueous extraction from Chondrus, Gigartina, and various Eucheuma species. Molecular weight Discrepancies in definitions. Native carrageenan reported to have average molecular weight of 1.5 x [10.sup.6] - 2 x [10.sup.7]; food-grade carrageenan reported as 100,000-800,000 or 200,000-400,000. Degraded carrageenan (poli- geenan) has average molecular weight of 20,000- 30,000; furcellaran has average molecular weight 20,000-80,000. Properties [lambda] and [kappa] combine easily with milk proteins to improve solubility and texture; serve as thickening agent, emulsifier, stabilizer. Synergistic effects With locust bean gum, increase in gel strength. Other hydrocolloids may also affect gel strength and cohesiveness. Concentration in 0.005-2.0% by weight. food products Major uses Milk products, processed meats, dietetic formulations, infant formula, toothpaste, cosmetics, skin preparations, pesticides, laxatives Table 2. Range of content of carrageenan in some commonly consumed foods. Percent carrageenan Food (g/100 g food) Bakery products 0.01-0.1 Chocolate milk 0.01-0.2 Cottage cheese 0.02-0.05 Frosting base mix 3-4 Ice cream, frozen custard, sherbets, etc. 0.01-0.05 Jams and jellies 0.5-1.2 Liquid coffee whitener 0.3 Pie filling 0.1-1.0 Pimento olive stuffing 2.0 Processed cheese 0.01-0.06 Processed meat or fish 0.2-1.0 Pudding (nondairy) 0.1-0.5 Relishes, pizza, barbecue sauces 0.2-0.5 Yogurt 0.2-0.5 Because manufacturing processes vary and there can be substitutions of one hydrocolloid for another, the content of carrageenan for any individual product may differ from these estimates. Unpublished manufacturers' data indicate that these content estimates for processed cheese, frozen dairy dessert, cottage cheese, and jams and jellies are significantly lower than current usage (4, 13, 14,47). Table 3. Experimental data related to intestinal effects of dietary carrageenan exposure. Experiment Type of carrageenan, Dose molecular weight Animal %CG (g/kg bw/day) 1. Undegraded (a,d) Rat 10 27.4 Undegraded (a,d) Rat 0.25 0.2 2.5 4 2. Undegraded Rat 0.5, 1.5, [iota], >100,000 5 (a,b) Degraded [iota], Rat 0.5, 1.5, 20,000 (a,b) 5 3. Degraded [iota], Guinea pig 2, 2.5, 5 30,000 (c) 4. Degraded, Guinea pig 3 5.8 20,000-30,000 (c) 5. Degraded (c) Guinea pig 1, 2, 3 2, 3, 4 6. Degraded (c) Rat ileal 0-1.5 g/L cell monolayers 7. Undegraded Rat 5 [lambda], 300,000 (a,b) 8. Degraded Rat, 5 [iota] (c) Guinea pig 5 9. Undegraded Rat 6 0.8 [kappa] (a,d) 10. Degraded Rabbit 1 [lambda] (a,b,c) 11. Degraded Mice 10 (a,b,c) 12. Degraded, Cultured 1 mg/10 mL (20,000-40,000), rat hepa- or and tocytes or 1 mg/100 mL Undegraded (a) intestinal mucosal cells 13. Undegraded Rat 0.5 0.15-0.25 [kappa], [lambda], and [iota] (e) 14. Degraded (c) Guinea pig 5 15. Degraded (c) Guinea pig 5 16. Degraded (a,d) Rat 10 17. Degraded, Rat 10 15 (20,000-40,000) (a,b,c,g) 18. Degraded Guinea pig 3 [kappa], [lambda], [iota] 19. Degraded (20,000-40,000) Rat 10 (a,b,e) 20. Degraded (c) Guinea 5 pig 21. Undegraded, Rat, 0.5, 2.5, 5 0.36, 2, 4 (800,000) hamster (largely [kappa]) 22. Degraded Rat 10 (a,b,c,g) 23. Degraded (c) Guinea 2 pig 24. Degraded (c) Guinea 5 pig 25. Degraded Rat 10, 5, 1 (20,000-40,000) (a,b) 5 1 or 5 26.Undegraded Rat 15 [lambda] (a,d) 27. [iota], Rat 0.5 (8,700-145,000) (e,f) Undegraded [kappa]/ Rat 5 [lambda], (186,000-214,000) [iota], Guinea pig 2 (5,000-145,000) [iota], Guinea pig 1 (5,000-145,000) [kappa] Guinea pig 1 (8,500-275,000) [lambda] Guinea pig 1 (8,500-275,000) Undegraded [kappa]/ [lambda] Rhesus 0.05, 0.2, 0.5 (185,000) monkey [iota] 0.2 28.[kappa] (c) Guinea 1 (314,000) pig (51,500) (8,500) [lambda], (275,000) (74,800) (20,800) [iota], (145,000) (107,000) (88,000) (39,000) (21,000) (8,700) (5,000) 29. Degraded Rhesus 2 [iota] (C16), monkey (20,000) Undegraded Rhesus 1 [kappa][lambda] monkey mixture, (800,000) 30. Degraded Rat 5 ([iota], C16), 7.5 g, (10,000-30,000) (a,d) then 5 g 31. Degraded (c,e) Rat 0.2, 0.5, 5 Guinea pig 0.25, 0.5 32. Degraded Guinea pig 2, 0.2, 0.02 ([iota], C16) (c) Guinea pig 2 Rat 5 Monkey 2 33. Degraded (c) Guinea pig 2, 5 1.7-3.3 34. Undegraded Pig 0.05, 0.2, 0.5 [kappa], (200,000) 35. Degraded Rhesus 0.5-2 0.7, 1.4, 2.9 (C16, [iota]), (c) monkey (20,000) Undegraded Rhesus 1 1.3 (largely [kappa]), monkey (800,000) 1-3 0.05-1.25 36. Undegraded Guinea pig 5 Rabbit 5 Degraded (c) Guinea pig 1, 2, 5 Rabbit 2 Degraded Humans 5-g dose Ferret 1.5 Squirrel 1.5 monkey Rabbit, 1.5 mouse Rat 1 Rat 5 Undegraded Rat 5 Hamster 5 37. Degraded Rat 5 6-10 (c16, [iota]), 0.5-5.0 (20,000-30,000) (a,b,e) 38. Undegraded Rhesus 1 ([kappa]:[lambda] monkey = 70:30), (800,000) (e) Degraded Rhesus 0.5, 1, 2 (C16, [iota]), monkey (20,000-30,000) (e) 39. Degraded (c) Guinea 5 [less than or pig equal to] 2 g 40. Degraded (a,b,c,g) Rat 5 41. Undegraded (c) Guinea 5 pig Degraded (c) Guinea 1 pig 42. Degraded (a,b,c) Rabbit 0.1, 1, 5 0.07, 0.8, 1.4 43. Degraded (c) Guinea 4-5 pig Degraded Rat [less than or equal to] 16.5 Degraded, Rat, 0.07-4 Undegraded mouse 44. Undegraded Guinea pig 1 [less than or equal to] 1.5 Degraded Guinea [less than [less than or pig or equal equal to] 2 to] 5 45. Degraded Guinea pig 0.1-5 Rabbit Rat Mouse Type of Experiment carrageenan, molecular weight Animal Duration Route 1. Undegraded (a,d) Rat 8 days, Jelly sacrificed at 30 days Undegraded (a,d) Rat 100 days Liquid 100 days Solid gel 2. Undegraded Rat [less than Diet [iota], >100,000 or equal to] (a,b) 91 days Degraded [iota], Rat [less than Diet 20,000 (a,b) or equal to] 91 days 3. Degraded [iota], Guinea pig 7 days Drink 30,000 (c) 4. Degraded, Guinea pig 2-3 days Drink 20,000-30,000 (c) 5. Degraded (c) Guinea pig 2 weeks Drink 6. Degraded (c) Rat ileal 19, 30, Media cell 54 hr monolayers 7. Undegraded Rat 4 weeks Diet [lambda], 300,000 (a,b) 8. Degraded Rat, 4 months Drink [iota] (c) Guinea pig 3 weeks Drink 9. Undegraded Rat 24 weeks diet [kappa] (a,d) 10. Degraded Rabbit 8 weeks, Drink [lambda] (a,b,c) 12 months, 28 months 11. Degraded Mice 10 days Drink (a,b,c) 12. Degraded, Cultured 20 hr (20,000-40,000), rat hepa- and tocytes or 2 hr Undegraded (a) intestinal mucosal cells 13. Undegraded Rat 90 days Drink [kappa], [lambda], and [iota] (e) 14. Degraded (c) Guinea pig 5 days Drink 15. Degraded (c) Guinea pig 14 days Drink 16. Degraded (a,d) Rat 2 weeks Diet 17. Degraded, Rat < 63 days Diet (20,000-40,000) (a,b,c,g) 18. Degraded Guinea pig 3 weeks Drink [kappa], [lambda], [iota] 19. Degraded 2, 6, 9 Diet (20,000-40,000) Rat months (a,b,e) sacrificed at 18 months 20. Degraded (c) Guinea [less than Drink pig or equal to] 28 days 21. Undegraded, Rat, Lifetime Diet (800,000) hamster (largely [kappa]) 22. Degraded Rat 1 day to 12 Diet (a,b,c,g) weeks, some sacrificed at 27 weeks 23. Degraded (c) Guinea 2 weeks Drink pig 24. Degraded (c) Guinea 21 days pig 25. Degraded Rat [less than Diet (20,000-40,000) (a,b) or equal to] 24 months 15 months drink 15 months Stomach tube 26.Undegraded Rat [less than Diet [lambda] (a,d) or equal to] 40 weeks 27. [iota], Rat 9 months Gavage (8,700-145,000) (e,f) Undegraded [kappa]/ Rat 13 weeks Diet [lambda], (186,000-214,000) [iota], Guinea pig 7-10 weeks Diet (5,000-145,000) [iota], Guinea pig 2-3 weeks Drink (5,000-145,000) [kappa] Guinea pig 2-3 weeks Drink (8,500-275,000) [lambda] Guinea pig 2-3 weeks Drink (8,500-275,000) Undegraded [kappa]/ [lambda] Rhesus Stomach tube (185,000) monkey [iota] Stomach tube 28.[kappa] (c) Guinea (314,000) pig 2 weeks Drink (51,500) 2 weeks Drink (8,500) 2 weeks Drink [lambda], (275,000) 2 weeks Drink (74,800) 2 weeks Drink (20,800) 2 weeks Drink [iota], (145,000) 2 weeks Drink 10 weeks Diet (107,000) 2 weeks Drink 10 weeks Diet (88,000) 2 weeks Drink (39,000) 2 weeks Drink 10 weeks Diet (21,000) 2 weeks Drink 10 weeks Diet (8,700) 2 weeks Drink 10 weeks Diet (5,000) 2 weeks Drink 29. Degraded Rhesus 10 weeks Drink [iota] (C16), monkey (20,000) Undegraded Rhesus 10 weeks Drink [kappa][lambda] monkey mixture, (800,000) 30. Degraded Rat [less than Drink, ([iota], C16), or equal to] diet (10,000-30,000) (a,d) 30 weeks 31. Degraded (c,e) Rat [less than Drink or equal to] 12 weeks Guinea pig [less than Drink or equal to] 4 weeks 32. Degraded Guinea pig 12 months, Drink ([iota], C16) (c) 10 months Guinea pig 3 months In milk Rat 3 months Drink Monkey 10 weeks Drink 33. Degraded (c) Guinea pig 30-44 days Drink 34. Undegraded Pig 83 days Jelly [kappa], (200,000) 35. Degraded Rhesus 7-14 weeks, Drink (C16, [iota]), (c) monkey then reco- (20,000) very for 20- 24 weeks for some before sacrificed Undegraded Rhesus 7-14 weeks, Drink (largely [kappa]), monkey then 11 (800,000) weeks reco- very [less than Tube or equal to] 12 weeks, after reco- very 36. Undegraded Guinea pig 1-45 days Diet Rabbit Diet Degraded (c) Guinea pig 1-45 days Drink Rabbit Drink Degraded Humans 10 days Diet Ferret 28 days Tube Squirrel 28 days Tube monkey Rabbit, 28 days Tube mouse Rat 56 days Drink Rat Undegraded Rat 56 days Diet Hamster 6 months Diet 37. Degraded Rat [greater Drink (c16, [iota]), than or equal to] 25 weeks (20,000-30,000) (a,b,e) 1-15 months Tube 38. Undegraded Rhesus 7-12 weeks Drink ([kappa]:[lambda] monkey = 70:30), (800,000) (e) Degraded Rhesus 7-12 weeks Drink (C16, [iota]), monkey (20,000-30,000) (e) 39. Degraded (c) Guinea 20-45 days Drink pig 40. Degraded (a,b,c,g) Rat 6 months Drink 41. Undegraded (c) Guinea 2-4 weeks Diet pig Degraded (c) Guinea 2-4 weeks Drink pig 42. Degraded (a,b,c) Rabbit 6-12 weeks Drink 43. Degraded (c) Guinea Drink pig Degraded Rat Drink Degraded, Rat, 28 days- Tube Undegraded mouse 6 months 44. Undegraded Guinea pig 20-30 days Drink Degraded Guinea 20-30 days Drink pig 45. Degraded Guinea pig 30 days- Drink Rabbit 1 year Drink Rat Drink Mouse Drink Experiment Effects Type of carrageenan, Additional Digestive/ molecular weight Animal exposure systemic 1. Undegraded (a,d) Rat AOM injection Less weight (5 mg/kg ip) gain with 2.5% CG Undegraded (a,d) Rat AOM injection (20 mg/kg ip) prior to CG 2. Undegraded Rat [iota], >100,000 (a,b) Degraded [iota], Rat 20,000 (a,b) 3. Degraded [iota], Guinea pig Eicosapen- 2% led to FOB+ 30,000 (c) taenoic acid at 7 days; 5% (300 mg/kg/day) led to 38% at 14 days mortality 4. Degraded, Guinea pig 20,000-30,000 (c) 5. Degraded (c) Guinea pig 6. Degraded (c) Rat ileal cell monolayers 7. Undegraded Rat [lambda], 300,000 (a,b) 8. Degraded Rat, [iota] (c) Guinea pig 9. Undegraded Rat 1,2-DMH [kappa] (a,d) (20 mg/kg bw) SC x 16 wks 10. Degraded Rabbit [lambda] (a,b,c) 11. Degraded Mice Bloody diarrhea (a,b,c) 12. Degraded, Cultured (20,000-40,000), rat hepa- and tocytes or Undegraded (a) intestinal mucosal cells 13. Undegraded Rat [kappa], [lambda], and [iota] (e) 14. Degraded (c) Guinea pig 15. Degraded (c) Guinea pig With ileo- transvers- ostomy 16. Degraded (a,d) Rat 1,2-DMH weekly injections for 15 weeks (10 mg/kg bw) with DCG vs. DMH alone 17. Degraded, Rat Germ-free vs. (20,000-40,000) conventional (a,b,c,g) gut flora 18. Degraded Guinea pig Diarrhea after [kappa], [lambda], 7 days. Mori- [iota] bund after 9 days of [iota] DCG. 19. Degraded Basal diet after (20,000-40,000) Rat DCG exposure (a,b,e) 20. Degraded (c) Guinea pig 21. Undegraded, Rat, Diarrhea in (800,000) hamster some (largely [kappa]) 22. Degraded Rat (a,b,c,g) 23. Degraded (c) Guinea Loose stools by pig 2 weeks 24. Degraded (c) Guinea 17/22 died pig by day 21 25. Degraded Rat (20,000-40,000) (a,b) 26.Undegraded Rat NMU 2 mg twice [lambda] (a,d) weekly rectally for 3 weeks; AOM (8 mg/kg bw) SC for 10 weeks; UCG with NMU, UCG with AOM 27. [iota], Rat (8,700-145,000) (e,f) Undegraded [kappa]/ Rat [lambda], (186,000-214,000) [iota], Guinea pig (5,000-145,000) [iota], Guinea pig (5,000-145,000) [kappa] Guinea pig (8,500-275,000) [lambda] Guinea pig (8,500-275,000) Undegraded [kappa]/ [lambda] Rhesus (185,000) monkey [iota] 28.[kappa] (c) Guinea (314,000) pig (51,500) (8,500) [lambda], (275,000) (74,800) (20,800) [iota], (145,000) (107,000) (88,000) (39,000) (21,000) (8,700) (5,000) 29. Degraded Rhesus [iota] (C16), monkey (20,000) Undegraded Rhesus [kappa][lambda] monkey mixture, (800,000) 30. Degraded Rat 1,2-DMH Watery, bloody ([iota], C16), (20 mg/kg) stools (10,000-30,000) (a,d) SC/wk 31. Degraded (c,e) Rat Severe diarrhea in 3 days with 5% Guinea pig Diarrhea 32. Degraded Guinea pig ([iota], C16) (c) Guinea pig Rat Monkey 33. Degraded (c) Guinea pig Trimethoprim/ Blood in stools Sulfame- thoxazole 34. Undegraded Pig [kappa], (200,000) 35. Degraded Rhesus Diarrhea, (C16, [iota]), (c) monkey hemorrhage (20,000) Undegraded Rhesus (largely [kappa]), monkey (800,000) 36. Undegraded Guinea pig Neomycin Diarrhea, Rabbit (0.1%) added hemorrhage Degraded (c) Guinea pig Rabbit Degraded Humans Ferret Squirrel monkey Rabbit, mouse Rat SI diarrhea Rat Diarrhea Undegraded Rat SI diarrhea Hamster Diarrhea 37. Degraded Rat FOB+ by (c16, [iota]), 3-7 days with > 5 g/kg/day; gross blood (20,000-30,000) (a,b,e) by 2-3 weeks 38. Undegraded Rhesus ([kappa]:[lambda] monkey = 70:30), (800,000) (e) Degraded Rhesus (C16, [iota]), monkey (20,000-30,000) (e) 39. Degraded (c) Guinea FOB+, pig diarrhea by 1 week 40. Degraded (a,b,c,g) Rat 41. Undegraded (c) Guinea pig Degraded (c) Guinea pig 42. Degraded (a,b,c) Rabbit Diarrhea, blood by day 7, weight loss 43. Degraded (c) Guinea pig Degraded Rat Degraded, Rat, Undegraded mouse 44. Undegraded Guinea pig FOB+ Degraded Guinea Diarrhea by pig 10 days, FOB+ 45. Degraded Guinea pig Weight loss in Rabbit guinea pig and Rat rabbit, not rat Mouse or mouse. Blood and mucous in stool. Type of Effects carrageenan, molecular weight Animal Histopathologic changes 1. Undegraded (a,d) Rat Undegraded (a,d) Rat 2. Undegraded Rat Epithelial cell loss, macrophage [iota], >100,000 infiltration, loss of crypts, (a,b) Degraded [iota], Rat 20,000 (a,b) 3. Degraded [iota], Guinea pig Cecal ulcerations; foamy 30,000 (c) macrophages; small epithelial ulcerations at 2 days. 4. Degraded, Guinea pig Microscopic mucosal changes from 20,000-30,000 (c) cecum to rectum; apparent macro- molecule absorption by colonic epithelium, macrophage infiltra- tion, macrophages with vacuoles. 5. Degraded (c) Guinea pig 100% had cecal ulceration after 3% for 4 days; crypt abscesses. 6. Degraded (c) Rat ileal cell monolayers 7. Undegraded Rat 3/8 had slight congestion [lambda], and erythema of distal colon. 300,000 (a,b) 8. Degraded Rat, Increased permeability to [iota] (c) Guinea pig ([sup.3] H) PEG-900; ulcerations in guinea pig, crypt abscesses, macrophage infiltration. 9. Undegraded Rat [kappa] (a,d) 10. Degraded Rabbit Ulcerative lesions at 8 wks; at [lambda] (a,b,c) 12 months had chronic inflammatory changes. 11. Degraded Mice Ulceration in proximal and distal (a,b,c) colon, with dilatation of cecum and ascending colon. 12. Degraded, Cultured (20,000-40,000), rat hepa- and tocytes or Undegraded (a) intestinal mucosal cells 13. Undegraded Rat CG able to penetrate intestine; [kappa], [lambda], CG in mesenteric lymph node, and [iota] (e) and macrophages of villus and lamina propria. 14. Degraded (c) Guinea pig Small, superficial ulcerations over mucosal surface of cecum (1-111 ulcerations/ [cm.sup.2]). 15. Degraded (c) Guinea pig Ulcerations in cecum and proximal colon in unoperated. Postproce- dure, crypt abscesses in rectum and ulcerations in distal colon and rectum. Macrophage infiltra- tion. 16. Degraded (a,d) Rat 17. Degraded, Rat Erosions; aggregates of foamy (20,000-40,000) metachromatic macrophages (a,b,c,g) in submucosa and lamina propria. 18. Degraded Guinea pig No cecal or colonic [kappa], [lambda], lesions seen. [iota] 19. Degraded CG in mucosa (20,000-40,000) Rat and RE system. (a,b,e) 20. Degraded (c) Guinea Cecal lesions after 24 hr; pig confluent ulcerations after 7 days. Macrophage infiltration. 21. Undegraded, Rat, No difference in (800,000) hamster ulcerations from control. (largely [kappa]) 22. Degraded Rat Superficial erosions (a,b,c,g) at anorectal junction at 24 hr; at 2 weeks, more proximal erosions. 23. Degraded (c) Guinea 100% with colonic ulcerations; pig 75% had over 200 ulcers. 24. Degraded (c) Guinea All had mucosal ulcerations pig from cecum to rectum by day 14. 25. Degraded Rat (20,000-40,000) (a,b) 26.Undegraded Rat [lambda] (a,d) 27. [iota], Rat Av MW of CG in liver was (8,700-145,000) (e,f) at 10,000; all CG in feces Undegraded [kappa]/ Rat had MW < 100,000. [lambda], (186,000-214,000) [iota], Guinea pig (5,000-145,000) [iota], Guinea pig (5,000-145,000) [kappa] Guinea pig (8,500-275,000) [lambda] Guinea pig (8,500-275,000) Undegraded [kappa]/ [lambda] Rhesus (185,000) monkey [iota] 28.[kappa] (c) Guinea Cecal ulceration not seen with (314,000) pig [kappa] or [lambda,]; [iota] (51,500) fractions of MW 21,000-107,000 (8,500) led to ulcerations of cecum, [lambda], crypt abscesses, and epithelial (275,000) thinning. [iota] fractions (74,800) absorbed and seen in vacuolated (20,800) macrophages. Intense lysosomal [iota], enzymatic activity in macrophages (145,000) of lamina propria. (107,000) (88,000) (39,000) (21,000) (8,700) (5,000) 29. Degraded Rhesus Macrophages given DCG had [iota] (C16), monkey fibrillar material and (20,000) vacuolations. Undegraded Rhesus Vacuolations seen with UCG. [kappa][lambda] monkey mixture, (800,000) 30. Degraded Rat Distal rectum transformed ([iota], C16), to stratified squamous by (10,000-30,000) (a,d) DMH with DCG. 31. Degraded (c,e) Rat DCG contained within macrophages of spleen, liver, kidney, small and large intestine; cecal Guinea pig and colonic ulcerations at 4 weeks. 32. Degraded Guinea pig 2% CG in water, but not in ([iota], C16) (c) milk, led to cecal ulceration Guinea pig in guinea pig. DCG in macro- phages of submucosal layer in guinea pigs, rats, and monkeys. Rat No cecal ulceration seen in Monkey rats or monkeys. 33. Degraded (c) Guinea pig Cecum and distal colon had ulcerations, crypt abscesses; enlarged cecal or colonic lymph nodes; more extensive ulceration with 5%; fewer lesions with antibiotic. Infiltration of foamy macrophages. 34. Undegraded Pig Focal irregularities without [kappa], ulcerations; thickened lamina (200,000) propria; macrophage infiltration. 35. Degraded Rhesus Ulcerations of colon; hypertrophy (C16, [iota]), (c) monkey of mesenteric lymph nodes and (20,000) granulomas; multiple crypt abscesses; dose effect present. Undegraded Rhesus Without colonic changes. (largely [kappa]), monkey (800,000) 36. Undegraded Guinea pig Multiple pinpoint cecal and Rabbit colonic ulcerations after 3-5 Degraded (c) Guinea pig weeks in guinea pig and rabbit. Rabbit Macrophages increased; inclusions and vacuoles in macrophages; granulomas seen. Neomycin did not affect incidence of ulcers or time of onset. Degraded Humans Patients had colon malignancy with colectomy planned to follow CG exposure, no ulcerations seen. Ferret No lesions seen. Squirrel No lesions seen. monkey Rabbit, mouse No lesions seen. Rat Rat Undegraded Rat Hamster 37. Degraded Rat Metachromatic material (c16, [iota]), thought to be CG found in RE cells of liver, spleen, lymph nodes, macrophages (20,000-30,000) (a,b,e) of lamina propria and submucosa. No cecal lesions. 38. Undegraded Rhesus No changes in liver. ([kappa]:[lambda] monkey = 70:30), (800,000) (e) Degraded Rhesus Membrane-bound vacuoles (C16, [iota]), monkey with fibrillar material in RE (20,000-30,000) (e) cells of liver. 39. Degraded (c) Guinea Multiple ulcers in cecum, colon, pig and rectum in 100% of animals by day 30. 40. Degraded (a,b,c,g) Rat Ulceration of cecum in 4/12, associated with stricture; marked glandular hyperplasia at ulcer margins. 41. Undegraded (c) Guinea Ulceration of mucosa as pig consequence of macrophage Degraded (c) Guinea accumulation in lamina propria, pig then submucosa. 42. Degraded (a,b,c) Rabbit Ulceration of colon in 100% of those fed 1%; 60% of those fed 0.1%. 43. Degraded (c) Guinea Mucosal erosions in cecum, pig rarely into colon in guinea pig; without erosion in rat or mouse. Degraded Rat Degraded, Rat, Undegraded mouse 44. Undegraded Guinea pig Multiple ulcerations of cecum; 80% had ulcerations. Crypt abscesses present; macrophages, with metachromatic material. Degraded Guinea 100% had ulcerations; ulceration pig extended into distal colon and rectum. 45. Degraded Guinea pig Hemorrhagic and ulcerative Rabbit lesions in cecum, colon, or Rat rectum in all four species; Mouse crypt abscesses present. Effects Type of carrageenan, Refe- molecular weight Animal Neoplastic changes rence 1. Undegraded (a,d) Rat UCG jelly (10% x 8 days) (50) did not initiate tumor. Undegraded (a,d) Rat UCG solid gel promoted growth of aberrant crypt foci (+15%; p = 0.019). 2. Undegraded Rat 5-Fold increase in (51) [iota], >100,000 thymidine kinase (a,b) activity in colon cells Degraded [iota], Rat with 5% UCG or DCG. 20,000 (a,b) 35-Fold increase in proliferating cells in upper third of crypt with DCG, 8-fold with UCG. 3. Degraded [iota], Guinea pig (52) 30,000 (c) 4. Degraded, Guinea pig (35) 20,000-30,000 (c) 5. Degraded (c) Guinea pig (53) 6. Degraded (c) Rat ileal Retarded cell growth (54) cell caused cell death; monolayers at 0.25g/L inhibited DNA synthesis by 20%. 7. Undegraded Rat 4-Fold increase in (55) [lambda], thymidine kinase 300,000 (a,b) activity in distal 12 cm of colonic mucosa. 8. Degraded Rat, (56) [iota] (c) Guinea pig 9. Undegraded Rat More tumors with (57) [kappa] (a,d) UCG than control diet (75% vs. 40%); also larger, more proximal tumors. 10. Degraded Rabbit At 28 months, focal (58) [lambda] (a,b,c) and severe glandular atypism; precancerous changes seen. 11. Degraded Mice 2-Fold increase in (59) (a,b,c) colonic epithelial cell proliferation; increase in labeling indices and extension of prolifera- tive compartment to upper third of crypt. 12. Degraded, Cultured DCG and UCG nonmuta- (60) (20,000-40,000), rat hepa- genic in Salmonella and tocytes or mutagenicity test; DCG Undegraded (a) intestinal nongenotoxic by DNA mucosal repair test. cells 13. Undegraded Rat (61) [kappa], [lambda], and [iota] (e) 14. Degraded (c) Guinea pig (62) 15. Degraded (c) Guinea pig (63) 16. Degraded (a,d) Rat Increase in tumors (64) of small intestine (50% vs. 25%) and colon (60% vs. 45%) with CG than occurred with DMH alone. 17. Degraded, Rat Squamous metaplasia (65) (20,000-40,000) from anorectal junction (a,b,c,g) to distal colon. 18. Degraded Guinea pig (66) [kappa], [lambda], [iota] 19. Degraded 100% incidence of (67) (20,000-40,000) Rat colorectal squamous (a,b,e) metaplasia that prog- ressed after DCG intake discontinued. 20. Degraded (c) Guinea (68) pig 21. Undegraded, Rat, Increased incidence (69) (800,000) hamster of benign mammary (largely [kappa]) tumors and testicular neoplasms (at 2.5% level) in rats only. 22. Degraded Rat Squamous metaplasia (70) (a,b,c,g) of rectal mucosa at 2 weeks; extended after no longer being fed CG. 23. Degraded (c) Guinea (34) pig 24. Degraded (c) Guinea (71) pig 25. Degraded Rat Squamous cell (72) (20,000-40,000) (a,b) carcinomas, adenocarcinomas, adenomas. 32% and fed 10% diet had tumors. 100% incidence of meta- plasia with 5% drink. 26.Undegraded Rat 100% had tumors with (73) [lambda] (a,d) AOM and UCG vs. 57% with AOM alone. 100% had tumors with NMU and UCG vs. 59% with NMU alone. 0 tumors in control, 7% tumors with UCG alone. UCG with AOM had 10-fold increase in number of tumors per rat. 27. [iota], Rat (74) (8,700-145,000) (e,f) Undegraded [kappa]/ Rat [lambda], (186,000-214,000) [iota], Guinea pig (5,000-145,000) [iota], Guinea pig (5,000-145,000) [kappa] Guinea pig (8,500-275,000) [lambda] Guinea pig (8,500-275,000) Undegraded [kappa]/ [lambda] Rhesus (185,000) monkey [iota] 28.[kappa] (c) Guinea (75) (314,000) pig (51,500) (8,500) [lambda], (275,000) (74,800) (20,800) [iota], (145,000) (107,000) (88,000) (39,000) (21,000) (8,700) (5,000) 29. Degraded Rhesus (76) [iota] (C16), monkey (20,000) Undegraded Rhesus [kappa][lambda] monkey mixture, (800,000) 30. Degraded Rat DMH with DCG- (77) ([iota], C16), induced proliferation (10,000-30,000) (a,d) of deep glandular areas; more poorly differentia- ted adenocarcinomas; more frequently found tumors of ascending and transverse colon with DMH and DCG. 31. Degraded (c,e) Rat (78) Guinea pig 32. Degraded Guinea pig (79) ([iota], C16) (c) Guinea pig Rat Monkey 33. Degraded (c) Guinea pig (80) 34. Undegraded Pig (81) [kappa], (200,000) 35. Degraded Rhesus (82) (C16, [iota]), (c) monkey (20,000) Undegraded Rhesus (largely [kappa]), monkey (800,000) 36. Undegraded Guinea pig (83) Rabbit Degraded (c) Guinea pig Rabbit Degraded Humans Ferret Squirrel monkey Rabbit, mouse Rat Rat Undegraded Rat Hamster 37. Degraded Rat Adenomatous (84) (c16, [iota]), and hyperplastic polyps in one rat. Squamous metaplasia (20,000-30,000) (a,b,e) of anorectal region and distal colon. 38. Undegraded Rhesus (85) ([kappa]:[lambda] monkey = 70:30), (800,000) (e) Degraded Rhesus (C16, [iota]), monkey (20,000-30,000) (e) 39. Degraded (c) Guinea (86) pig 40. Degraded (a,b,c,g) Rat (87) 41. Undegraded (c) Guinea (88) pig Degraded (c) Guinea pig 42. Degraded (a,b,c) Rabbit Hyperplastic 89, mucosal changes, 90) polypoidal lesions. 43. Degraded (c) Guinea (91) pig Degraded Rat Degraded, Rat, Undegraded mouse 44. Undegraded Guinea pig (92) Degraded Guinea pig 45. Degraded Guinea pig (93) Rabbit Rat Mouse Abbreviations: AOM, azoxymethane; bw, body weight, CG,carrageenan; DCG, degraded carrageenan; DMH, dimethylhydrazine; FOB, fecal occult blood; ip, intraperitoneal; NMU, nitrosomethylurea; PEG, polyethylene glycol; SC, subcutaneous; SI, slight; tube, gastric intubation; UCG, undegraded carrageenan. (a) Studies are associated with neoplastic changes, unlike studies predominantly demonstrating intestinal ulcerations. (b) Increased proliferation or neoplasm and carrageenan alone. (c) Ulcerations and carrageenan alone. (d) Neoplasms in which carrageenan promoted carcinogenesis. (e) Studies with uptake to lymph node or other site. (f) Study demonstrating breakdown to lower molecular weight. (g) Studies demonstrating ulcerations in rat using degraded carrageenan. Table 4. Proposed mechanism for effects of carrageenan (9,10,35,67,72,75,76,79,84-86,88,98,102,105,107-110,114,115). Site Effect Intestinal lumen Ingested carrageenan can undergo acid hydrolysis in stomach possible breakdown by intestinal bacteria. Intestinal Take up degraded carrageenan, as indicated by epithelial cells metachromatic staining from cecum to rectum. Vacuoles observed to contain metachromatic material. Epithelial cells may undergo lysis from effect of lysosomal disruption producing erosions. Inflammatory Polymorphonuclear cells and macrophages infiltrate infiltrate to site of intestinal inflammation. Macrophages have metachromatic staining associated with uptake of degraded carrageenan. Lysosomal vacuolation occurs as well as lysosomal disruption with release of intracellular enzymes from macrophage destruction, leading to intestinal ulcerations. Process of chronic inflammation, as with ulcerative colitis. Macrophage Macrophages may circulate and may lead to circulation extraintestinal effects related to carrageenan. Table 5. Experimental evidence for presence of low molecular weight carrageenan in food-grade carrageenan and production of low molecular weight carrageenan by acid hydrolysis or by bacteria. (9,10,36-40). Degraded carrageenan in food-grade carrageenan 25% of total carrageenans in eight food-grade [kappa]-carrageenans had MW < 100,000 9% of total carrageenan in eight food-grade [kappa]-carrageenans had MW < 50,000 Production of degraded carrageenan by acid hydrolysis of food-grade carrageenan In simulated gastric fluid (including pepsin and HCL}, [kappa]- carrageenan at pH 1.2, 37 [degrees] C for 1 hr leads to 17% degraded carrageenan with MW < 20,000 for 2 hr leads to 25% with MW < 20,000 In simulated gastric fluid (including pepsin and HCL), [kappa]- carrageenan at pH 1.9, 37 [degrees] C for 1 hr leads to 8% with MW < 20,000 for 2 hr leads to 10% with MW < 20,000 [kappa]-carrageenan in solution at pH 1.0, 37 [degrees] C, for 6 hours, leads to 25% with MW < 20,000 [iota]-carrageenan in solution at pH 1.0, 37 [degrees] C, for 6 hours, leads to 10% with MW < 25,000 Hydrolysis of carrageenan by bacterial carrageenases [kappa]- and [iota]-carrageenase from cell-free supernatant from culture of Cytophaga genus [kappa]-carrageenase isolated from cell-free medium of cultured Pseudomonas carrageenovora [lambda]-carrageenase from cell-free medium of Pseudomonas carrageenovora cultures MW, molecular weight.
REFERENCES AND NOTES
(1.) Ries LAG, Kosary CL, Hankey BF, Miller BA; Clegg L, Edwards BK. SEER Cancer Statistics Review 1973-1996. Bethesda, MD:National Cancer Institute, 1999.
(2.) Schottenfeld D, Winawer SJ. Cancers of the large intestine. In: Cancer Epidemiology and Prevention (Schottenfeld D, Fraumeni J, eds). 2nd ed. New York:Oxford University Press, 1996;813-840.
(3.) Schatzkin A. Available: http://rex.nci.nih.gov/ NCI_Pub_Interface/raterisk/risks129.html [cited 6 October 2000].
(4.) IARC. IARC Working Group on the Evaluation or the Carcinogenic Risk of Chemicals to Humans. Carragaenan. IARC Monogr Eval Carcinog Risk Hum 31:79-94 (1983).
(5.) National Research Council. Carcinogens and Anti-carcinogens in the Human Diet. Washington, DC:National Academy Press, 1996;398.
(6.) Marcus R, Watt J. Danger of carrageenan in foods and [Letter]. Lancet 1:338 (1981).
(7.) Marcus R, Watt J. Potential hazards of carrageenan [Letter]. Lancet 1:802-603 (1980).
(8.) Marcus R. Harmful effects of carrageenan fed to animals. Cancer Detect Prey 4:129-134 (1981).
(9.) Ekstrom L-G. Molecular weight distribution and the behavior of kappa-carrageenan on hydrolysis. Carbohydr Res 135:283-289 (1985).
(10.) Ekstrom L-G, Kuivinen J, Johansson G. Molecular weight distribution and hydrolysis behavior of carrageenans. Carbohydr Res 116:89-94 (1983).
(11.) Yu G, Ioanoviciu AS, Sikkander SA, Thanawiroon C, Toida T, Tobacman J, Linhardt RJ. Unpublished data.
(12.) Klose RE, Glicksman M. Gums. In: Handbook of Food Additives (Furia TE, ed). Cleveland, OH:The Chemical Rubber Co., 1968;313-375.
(13.) Towle GA. Carrageenan. In: Industrial Gums: Polysaccharides and Their Derivatives (Whistler RL, ed). New York:Academic Press, Inc., 1973;84-109.
(14.) Moirano AL. Sulfated seaweed polysaccharides. In: Food Colloids (Graham HD, ed). Westport, CT:AVI Publishing Co., 1977;347-381.
(15.) Daniel JR, Voragen ACJ, Pilnik W. Starch and other polysaccharides. In: Ullmann's Encyclopedia of Industrial Chemistry, Vol A 25 (Elvers B, Hawkins S, Russey W, ads). New York:VCH Verlagsgesellschaft, 1994;21-62.
(16.) Substances that are generally recognized as safe. Fed Reg 21:9368-9370.
(17.) Food and Drugs: Food Additives. 21 C.F.R. 121.101,121.1063,121.1066,121.1067,121.1069,1969.
(18.) Proposed Revision of Food Additive Regulations and Deletion of Chondrus Extract (Carrageenin) from Generally Regarded as Safe (GRAS) List. 37 Fed Reg 15434.
(19.) Informatics, Inc. Carrageenan. Arlington, VA:National Technical and Information Service, 1972;1-68.
(20.) Nicklin S, Miller K. Intestinal uptake and immunological effects of carrageenan--current concepts. Food Addit Contain 6(4):425-436 (1989).
(21.) Food and Nutrition Board, National Research Council. Estimating Distribution of Daily Intakes of Chondrus Extract (Carrageenan): Committee on GRAS List Survey--Phase III. Appendix C. Washington, DC:National Academy of Sciences, 1976;1-7.
(22.) Stanicoff DJ, Renn DW. Physiological effects of carrageenan. In: ACS Symposium Series (15): Physiological Effects of Food Carbohydrates (Gould RF, ed). Washington, DC:American Chemical Society, 1975; 282-295.
(23.) Pintauro SJ, Gilbert SW. The effects of carrageenan on drug-metabolizing enzyme system activities in the guinea-pig. Food Chem Toxicol 28:807-611 (1990).
(24.) Carrageenan, Salts of Carrageenan and Chondrus Extract (Carrageenin); Withdrawal of Proposal and Termination of Rulemaking Proceeding. Fed Reg 44:40343-40345.
(25.) International Food Additives Council and FMC Corporation-Marine Colloids Division, filing of Food Additive Petitions; Hercules, Inc.; Notice of Receipt of Citizen Petition; Request for Comments. Fed Reg 57:49483-49485.
(26.) National Research Council. Food Chemical Codex. 2nd ed, suppl 2. Washington, DC:National Academy of Science, 1975.
(27.) National Research Council. Food Chemical Codex. 4th ed. Washington, DC:National Academy of Science, 1996.
(28.) Tong H-K, Lee K-H, Wong H-A. The molecular weight and viscosity of the water-soluble polysaccharide(s) from Eucheuma spinosum. Carbohydr Res 81:1-6 (1980).
(29.) Weiner ML. Toxicological properties of carrageenan. Agents Actions 32(1/2):48-51 (1991).
(30.) Food and Drugs: Food Additives Permitted for Direct Addition to Food for Human Consumption. 21 C.F.R. 172.620,172.626,172.655,172.660, 2000.
(31.) Food and Drugs: Substances Generally Regarded as Safe. 21 C.F.R. 182.7255,1999.
(32.) Food and Drugs: New Drugs. 21 C.F.R. 310.545, 1999.
(33.) Food and Drugs: 21 C.F.R. 133.178, 133.179, 136.110, 139.121,139.121,139.122, 150.141,150.161,176.170 (2000).
(34.) Watt J, McLean C, Marcus R. Degradation of carrageenan for the experimental production of ulcers in the colon. J Pharm Pharmacol 31:645--646 (1979).
(35.) Marcus SN, Marcus AJ, Marcus R, Ewen SWB, Watt J. The pre-ulcerative phase of carrageenan-induced colonic ulceration in the guinea-pig. Int J Exp Pathol 73:515-526 (1992).
(36.) Sarwar G, Matoyoshi S, Oda H. Purification of a [kappa]-carrageenase from marine cytophaga species. Microbiol Immunol 31:869-877 (1987).
(37.) Weigl J, Yaphe W. The enzymic hydrolysis of carrageenan by pseudomonas carrageenovora: purification of a [kappa]-carrageenase. Can J Microbiol 12:939-947 (1986).
(38.) Potin P, Sanseau A, LeGall Y, Rochas C, Bloareg B. Purification and characterization of a new [kappa]-carrageenase from a marine cytophaga-like bacterium. Eur J Biochem 201:241-247 (1991).
(39.) McLean MW, Williamson FB. [kappa]-Carrageenase from Pseudomonas carrageenovora. Eur J Biochem 93:553-558 (1979).
(40.) Johnston KH, McCandless EL. Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of "lambda carrageenases" from Pseudomonas carrageenovora. Can J Microbiol 19(7):779-788 (1973).
(41.) Friedman LJ, Greenwald CG. Food additives. In: Encyclopedia of Chemical Technology, Vol 11 (Howe-Grant M, ed). 4th ed. New York:John Wiley & Sons, 1994;805-833.
(42.) Meer WA. Plant hydrocolloids. In: Food Colloids (Graham HD, ed). Westport, CT:AVI Publishing Company, Inc., 1977;522-539.
(43.) Food and Nutrition Board, National Research Council. The 1977 Survey of Industry on the Use of Food Additives: Committee on GRAS List Survey-Phase III. Part 3. PB 80-113418. Washington, DC:National Academy of Sciences, 1979.
(44.) Anderson W. Carrageenan: structure and biological activity. Can J Pharm Sci 2:81-90 (1967).
(45.) Comite "Additifs Alimentaires" du CNERNA. Toxicological evaluation of carrageenans. 10-Conclusions: acquired knowledges and problems requiring further researches. Sciences des aliments 4:429-438.
(46.) Will R, Zuanich J, DeBoo A, Ishikawa Y. Water-soluble polymers. Menlo Park, CA:Chemical Economics Handbook - SRI International, 1999;582.0000E-582.0003V.
(47.) Piculell L Gelling carrageenans. In: Food Polysaccharides and Their Applications. New York:Marcel Dekker, Inc., 1995;205-244.
(48.) Food and Nutrition Board, National Research Council. 1977 Survey of Industry on the Use of Food Additives. Summarized Data: Committee on GRAS List Survey-Phase III. Washington, DC:National Academy of Sciences, 1979;978-987.
(49.) Food Protection Committee, Food and Nutrition Board, National Research Council. Chemicals Used in Food Processing. Publication 1274. Washington, DC:National Academy of Sciences, 1965;31-34.
(50.) Corpet DE, Tache S, Preclaire M. Carrageenan given as a jelly, does not initiate, but promotes the growth of aberrant crypt foci in the rat colon. Cancer Lett 114:53-55 (1997).
(51.) Wilcox DK, Higgins J, Bertram TA. Colonic epithelial cell proliferation in a rat model of nongenotoxin-induced colonic neoplasia. Lab Invest 67:405-411 (1992).
(52.) Kitsukawa Y, Saito H, Suzuki Y, Kasanuki J, Tamura Y, Yoshida S. Effect of ingestion of eicosapentaenoic acid ethyl ester on carrageenan-induced colitis in guinea pigs. Gastroenterology 102:1859-1866 (1992).
(53.) Marcus A J, Marcus SN, Marcus R, Watt J. Rapid production of ulcerative disease of the colon in newly-weaned guinea-pigs by degraded carrageenan. J Pharm Pharmacol 41:423-426 (1989).
(54.) Ling [kappa]-Y, Bhalla D, Hollander D. Mechanisms of carrageenan injury of IEC18 small intestinal epithelial cell monolayers. Gastroenterology 95:1487-1495 (1988).
(55.) Calvert RJ, Reicks M. Alterations in colonic thymidine kinase enzyme activity induced by consumption of various dietary fibers. Proc Soc Exp Biol Med 189:45-51 (1988).
(56.) Delahunty T, Recher L, Hollander D. Intestinal permeability changes in rodents: a possible mechanism for degraded carrageenan-induced colitis. Food Chem Toxicol 25:113-118 (1987).
(57.) Arakawa S, Okumua M, Yamada S, Ito M, Tejima S. Enhancing effect of carrageenan on the induction of rat colonic tumors by 1,2-dimethylhydrazine and its relation to [beta]-glucuronidase activities in feces and other tissues. J Nutr Sci Vitaminol (Tokyo) 32:481-485 (1986).
(58.) Kitano A, Matsumoto T, Hiki M, Hashimura H, Yoshiyasu K, Okawa K, Kuwajima S, Kobayashi K. Epithelial dysplasia of the rabbit colon induced by degraded carrageenan. Cancer Res 46:1374-1376 (1986).
(59.) Fath RB, Deschner EE, Winawer SJ, Dworkin BM. Degraded carrageenan-induced colitis in C[F.sub.1] mice. Digestion 29:197-203 (1984).
(60.) Mori H, Ohbayashi F, Hirono I, Shimada T, Williams GM. Absence of genotoxicity of the carcinogenic sulfated polysaccharide carrageenan and dextran sulfate in mammalian DNA repair and bacterial mutagenicity assays. Nutr Cancer 6:92-97 (1984).
(61.) Nicklin S, Miller K. Effect of orally administered food-grade carrageenans on antibody-mediated and cell-mediated immunity in the inbred rat. Food Chem Toxicol 22:615-621 (1984).
(62.) Jensen BH, Andersen JO, Poulsen SS, Olsen PS, Rasmussen SN, Hansen SH, Hvidberg DF. The prophylactic effect of 5-aminosalicylic acid and salazosulphapyridine on degraded-carrageenan-induced colitis in guinea pigs. Scand J Gastroenterol 19:299-303 (1984).
(63.) Olsen PS, Kirkegaard P, Poulsen SS. The effect of ileotransversostomy on carrageenan-induced colitis in guinea pig. Scand J Gastroenterol 18:407-410 (1983).
(64.) Kawaura A, Shibata M, Togei K, Otsuka H. Effect of dietary degraded carrageenan on intestinal carcinogenesis in rats treated with 1,2-dimethylhydrazine dihydrochloride. Tokushima J Exp Med 29:125-129 (1982).
(65.) Hirono I, Sumi Y, Kuhara K, Miyakawa M. Effect of degraded carrageenan on the intestine in germfree rats. Toxicol Lett 8:207-212 (1981).
(66.) Norris AA, Lewis AJ, Zeitlin IJ. Inability of degraded carrageenan fractions to induce inflammatory bowel ulceration in the guinea pig. J Pharm Pharmacol 33:612-613 (1981).
(67.) Oohashi Y, Ishioka TT, Wakabayashi K, Kuwabara N. A study of carcinogenesis induced by degraded carrageenan arising from squamous metaplasia of the rat colorectum. Cancer Lett 14:267-272 (1981).
(68.) Olsen PS, Poulsen SS. Stereomicroscopic and histologic changes in the colon of guinea pigs fed degraded carrageenan. Acta Pathol Microbiol Scand Sect A 88:135-141 (1980).
(69.) Rustia M, Shubik P, Patil K. Lifespan carcinogenicity tests with native carrageenan in rats and hamsters. Cancer Lett 11:1-10 (1980).
(70.) Oohashi Y, Kitamura S, Wakabayashi K, Kuwabara N, Fukuda Y. Irreversibility of degraded carrageenan-induced colorectal squamous metaplasia in rats. Gann 70:391-392 (1979).
(71.) Onderdonk AB, Hermos JA, Dzink JL, Bartlett JG. Protective effect of metronidazole in experimental ulcerative colitis. Gastroenterology 74:521-526 (1978).
(72.) Wakabayashi K, Inagaki T, Fujimoto Y, Fukuda Y. Induction by degraded carrageenan of colorectal tumors in rats. Cancer Lett 4:171-176 (1978).
(73.) Watanabe K, Reddy BS, Wong CQ, Weisburger JH. Effect of dietary undegraded carrageenan on colon carcinogenesis in F344 rats treated with azoxymethane or methylnitrosourea. Cancer Res 38:4427-4430 (1978).
(74.) Pittman KA, Golberg L, Coulston F. Carrageenan: the effect of molecular weight and polymer type on its uptake, excretion and degradation in animals. Food Cosmet Toxicol 14:85--93 (1976).
(75.) Engster M, Abraham R. Cecal response to different molecular weights and types of carrageenan in the guinea pig. Toxicol Appl Pharmacol 38:265-282 (1976).
(76.) Mankes R, Abraham R. Lysosomal dysfunction in colonic submucosal macrophages of rhesus monkeys caused by degraded iota carrageenan. Proc Soc Exp Biol Med 150:166-170 (1975).
(77.) Iatropoulos MJ, Golberg L, Coulston L. Intestinal carcinogenesis in rats using 1,2-dimethylhydrazine with or without degraded carrageenan. Exp Mol Pathol 23:386--401 (1975).
(78.) Grasso P, Gangolli SD, Butterworth KR, Wright MG. Studies on degraded carrageenan in rats and guinea-pigs. Food Cosmet Toxicol 13:195-201 (1975).
(79.) Abraham R, Fabian RJ, Golberg MB, Coulston F. Role of lysosomes in carrageenan-induced cecal ulceration. Gastroenterology 67:1169-1181 (1974).
(80.) Van der Waaif D, Cohen BJ, Anver MR. Mitigation of experimental inflammatory bowel disease in guinea pigs by selective elimination of the aerobic gram-negative intestinal microflora. Gastroenterology 67:460-472 (1974).
(81.) Poulsen E. Short-term peroral toxicity of undegraded carrageenan in pigs. Food Cosmet Toxicol 11:219-227 (1973).
(82.) Benitz [kappa]-F, Golberg L, Coulston F. Intestinal effects of carrageenans in the rhesus monkey. Food Cosmet Toxicol 11:565-575 (1973).
(83.) Grasso P, Sharratt M, Carpanini FMB, Gangolli SD. Studies on carrageenan and large-bowel ulceration in mammals. Food Cosmet Toxicol 11:555-564 (1973).
(84.) Fabian RJ, Abraham R, Coulston F, Golberg L. Carrageenan-induced squamous metaplasia of the rectal mucosa in the rat. Gastroenterology 65:265-276 (1973).
(85.) Abraham R, Golberg L, Coulston F. Uptake and storage of degraded carrageenan in lysosomes of reticuloendothelial cells of the rhesus monkey. Exp Mol Pathol 17:77-93 (1972).
(86.) Watt J, Marcus R. Carrageenan-induced ulceration of the large intestine in the guinea pig. Gut 12:164-171 (1971).
(87.) Marcus R, Watt J. Colonic ulceration in young rats fed degraded carrageenan. Lancet 2:765-766 (1971).
(88.) Sharratt M, Grasso P, Carpanini F, Gangolli SD. Carrageenan ulceration as a model for human ulcerative colitis. Lancet 2:932 (1970).
(89.) Watt J, Marcus R. Ulcerative colitis in rabbits fed degraded carrageenan. J Pharm Pharmacol 22:130-131 (1970).
(90.) Watt J, Marcus R. Hyperplastic mucosal changes in the rabbit colon produced by degraded carrageenin. Gastroenterology 59:760-768 (1970).
(91.) Maillet M, Bonfils S, Lister RE. Carrageenan: effects in animals. Lancet 2:414-415 (1970).
(92.) Watt J, Marcus R. Ulcerative colitis in the guinea-pig caused by seaweed extract. J Pharm Pharmacol 21:187S-188S (1969).
(93.) Marcus R, Watt J. Seaweeds and ulcerative colitis in laboratory animals. Lancet 2:489-490 (1969).
(94.) Onderdonk AB. The carrageenan model for experimental ulcerative colitis. Prog Clin Biol Res 186:237-245 (1985).
(95.) Ottet NK. On animal models for inflammatory bowel disease. Gastroenterology 62:1269-1272 (1972).
(96.) Watt J, Marcus R. Progress report: Experimental ulcerative disease of the colon in animals. Gut 14:506-510 (1973).
(97.) Sharratt M, Grasso P, Carpanini F, Gangolli SD. Carrageenan ulceration as a model for human ulcerative colitis. Lancet 1:192-193 (1971).
(98.) Mottet NK. On animal models for inflammatory bowel disease. Gastroenterology 62:1269-1271 (1971).
(99.) Kim H-S, Berstad A. Experimental colitis in animal models. Scand J Gastroenterol 27:529-537 (1992).
(100.) Watt J, Marcus SN, Marcus AJ. The comparative prophylactic effects of sulfasalazine, prednisolone, and azathioprine in experimental ulceration. J Pharm Pharmacol 32:873-874 (1980).
(101.) Kitano A, Matsumoto T, Oshitani N, Nakagawa M, Yasuda K, Watanabe Y, Tomobuchi M, Obayashi M, Tabata A, Fukushima R, et al. Distribution and anti-inflammatory effect of mesalazine on carrageenan-induced colitis in the rabbit. Clin Exp Pharmacol Physiol 23:305-309 (1996).
(102.) Ishioka T, Kuwabara N, Oohashi Y, Wakabayashi K. Induction of colorectal tumors in rats by sulfated polysaccharides. CRC Crit Rev Toxicol 17:215-244 (1987).
(103.) Gangolli SD, Wright MG, Grasso P. Identification of carrageenan in mammalian tissues: an analytical and histochemical study. Histochem J 5:37-48 (1973).
(104.) Pipy B. 9-Carraghenanes et macrophages. Sciences des aliments 4:415-428 (1984).
(105.) Catanzaro PJ, Schwartz HJ, Graham RD. Spectrum and possible mechanism of carrageenan cytotoxicity. Am J Pathol 64:387-404 (1971).
(106.) Thomson AW, Fowler EF. Carrageenan: a review of its effect on the immune system. Agents Actions 1:265-273 (1981).
(107.) Kolodny EW, Fluharty AL. Metachromatic leukodystrophy and multiple sulfatase deficiency: sulfatide lipidosis. In: The Metabolic and Molecular Bases of Inherited Diseases (Scriver CR, AL Beaudet AL, Sly WS, Valle D, ads). 7th ed. New York: McGraw-Hill, Inc., 1995; 2693-2739.
(108.) Ballabio A, Shapiro LJ. Steroid sulfatase deficiency and X-linked ichthyosis. In: The Metabolic and Molecular Bases of Inherited Diseases (Scriver CR, Beaudet AL, Sly WS, Valle D, eds) 7th ed. New York: McGraw-Hill, Inc., 1995;2999-3022.
(109.) Cotran RS, Kumar V, Robbins SL, Schoen FJ. Genetic diseases. Robbins' Pathological Basis of Disease. 5th ed. Philadelphia:W.B. Saunders Company, 1994;123-171.
(110.) Muenzer J. Mucopolysaccharidoses. Adv Pediatr 33:269-302 (1986).
(111.) Corpet DE. Toxicological evaluation of carrageenans. 5-Dietary carrageenans and intestinal microflora. Sciences des aliments 4:367-374 (1984).
(112.) Michel C, Macfarlane GT. Digestive fates of soluble polysaccharides from marine macroalgae: involvement of the colonic microflora and physiological consequences for the host. J Appl Bacteriol 1996;80:349-369 (1996).
(113.) Gibson GR, Macfarlane S, Cummings JH. The fermentability of polysaccharides by mixed human faecal bacteria in relation to their suitability as [kappa]-forming laxatives. Lett Appl Microbiol 11:251-254 (1990).
(114.) Roediger WEW, Duncan A, Kapaniris O, Millard S. Reducing sulfur compounds of the colon impair colonocytes nutrition: implications for ulcerative colitis. Gastroenterology 104:802-809 (1993).
(115.) Richardson CJ, Magee EAM, Cummings JH. A new method for the determination of sulphide in gastrointestinal contents and whole blood by microdistillation and ion chromatography. Clin Chim Acta 293:115-125 (2000).
(116.) Babidge W, Millard S, Roediger W. Sulfides impair short chain fatty acid beta-oxidation at acyl-CoA dehydrogenase level in colonocytes: implications for ulcerative colitis. Mol Cell Biochem 181:117-124 (1998).
(117.) Toscani A, Soprano DR, Soprano KJ. Molecular analysis of sodium butyrate-induced growth arrest. Oncogene Res 3:223-238 (1998).
(118.) Glinghammar B, Holmberg K, Rafter J. Effects of colonic lumenal components on AP-1 dependent gene transcription in cultured human colon carcinoma cells. Carcinogenesis 20:969-976 (1999).
(119.) Salyers AA, West SHE, Vercelotti JR, Wilkins TD. Fermentation of mucins and plant polysacchairds by anerobic bacteria from the human colon. Appl Environ Microbiol 334:529-533 (1977).
(120.) Di Rosa M. Review: Biological properties of carrageenan. J Pharm Pharmacol 24:89-102 (1972).
(121.) Hoffman R. Carrageenans inhibit growth-factor binding. Biochem J 289:331-334 (1993).
(122.) Cochran FR, Baxter CS. Macrophage-mediated suppression of T-lymphocyte proliferation induced by oral carrageenan administration. Immunology 53:221-227 (1994).
(123.) Thomson AW, Fowler EF. Potentiation of tumor growth by carrageenan. Transplantation 24:397-400 (1977).
(124.) Carlucci MJ, Pujol CA, Ciancia M, Noseda MD, Matulewicz MC, Damonte EB, Cerezo AS. Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity. Int J Biol Macromol 20:97-105 (1997).
(125.) Yamada T, Ogano A, Saito T, Watanabe J, Uchiyama H, Nakagawa Y. Preparation and anti-HIV activity of low-molecular-weight carrageenans and their sulfated derivatives. Carbohydr Polym 32:51-55 (1997).
(126.) Pearce-Pratt R, Phillips DM. Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1. Biol Reprod 54:173-182 (1996).
(127.) Zaretzky FR, Pearce-Pratt R, Phillips DM. Sulfated polyanions block Chlamydia trachomatis infection of cervix-derived human epithelia. Infect Immun 63:3520-3526 (1995).
(128.) Hoffman R, Burns WW, Paper DH. Selective inhibition of cell proliferation and DNA synthesis by the polysulphated carbohydrate ??-carrageenan. Cancer Chemother Pharmacol 36:325-334 (1995).
(129.) Coombe DR, Parish CR, Ramshaw IA, Snowden JM. Analysis of the inhibition of tumour metastasis by sulphated polysaccharides. Int J Cancer 39:82-88 (1987).
(130.) Tobacman JK. Filament disassembly and loss of mammary myoepithelial cells after exposure to lambda-carrageenan. Cancer Res 57:2823-2826 (1997).
(131.) Tobacman JK, Walters K. Carrageenan exposure leads to mammary myoepithelial cell development of unusual intracellular inclusions. Proc Am Assoc Cancer Res 39:4722 (1999).
(132.) Cater DB. The carcinogenic action of carrageenin in rats. Br J Cancer 15:607-614 (1961).
(133.) Hopkins J. Carcinogenicity of carrageenan. Food Cosmet Toxicol 19:779-788 (1981).
(134.) Dyrset N, Lystad KQ, Levine DW. Development of a fermentation process for production of a kappa-carrageenase from Pseudomonas carrageenovora. Enzyme Microb Technol 20(6):418-423 (1997).
(135.) Irvine EJ, Farrokhyar F, Swarbrick ET. A critical review of epidemiological studies in inflammatory bowel disease. Scand J Gastroenterol 36(1):2-15 (2001).
(136.) Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide, Version 1.0. IARCCancerBase No. 5. Lyon:IARC Press, 2001. Limited version available: http://www-dep.iarc.fr/cgibin/exe-globom.exe [cited 2 March 2001].
(137.) Gold LS, Slone TH, Manley NB, Garfinkel GB, Rohrbach L, Ames BN. Carcinogenic potency database. In: Handbook of Carcinogenic Potency and Genotoxicity Databases (Gold LS, Zeiger E, ads). New York:CRC Press, Inc., 1997;116-117.
(138.) Gold LS, Slone TH, Ames BN. Summary of carcingogenic potency database by chemical. In: Handbook of Carcinogenic Potency and Genotoxicity Databases (Gold LS, Zeiger E, ads). New York:CRC Press, Inc., 1997;629.
(139.) Food Additives Amendment of 1958. Public Law 85-929, 72 Stat. 1784.
(140.) Food Quality Protection Act of 1996. Public Law 104-170, 110 Stat. 1489.
Joanne K. Tobacman College of Medicine, University of Iowa, Iowa City, Iowa, USA
Address correspondence to J.K. Tobacman, Department of Internal Medicine, University of Iowa Health Care, 200 Hawkins Drive, Iowa City, Iowa 52242-1081, USA. Telephone: (319) 356-3702. Fax: (319) 356-3086. E-mail: joanne-tobacman@ uiowa.edu
Received 17 January 2001; accepted 17 March 2001.