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Review of harmful gastrointestinal effects of carrageenan in animal experiments. (Research Review).

In this article I review the association between exposure to carrageenan and the occurrence of colonic ulcerations and gastrointestinal neoplasms in animal models. Although the International Agency for Research on Cancer in 1982 identified sufficient evidence for the carcinogenicity of degraded carrageenan in animals to regard it as posing a carcinogenic risk to humans, carrageenan is still used widely as a thickener, stabilizer, and texturizer in a variety of processed foods prevalent in the Western diet. I reviewed experimental data pertaining to carrageenan's effects with particular attention to the occurrence of ulcerations and neoplasms in association with exposure to carrageenan. In addition, I reviewed from established sources mechanisms for production of degraded carrageenan from undegraded or native carrageenan and data with regard to carrageenan intake. Review of these data demonstrated that exposure to undegraded as well as to degraded carrageenan was associated with the occurrence of intestinal ulcerations and neoplasms. This association may be attributed to contamination of undegraded carrageenan by components of low molecular weight, spontaneous metabolism of undegraded carrageenan by acid hydrolysis under conditions of normal digestion, or the interactions with intestinal bacteria. Although in 1972, the U.S. Food and Drug Administration considered restricting dietary carrageenan to an average molecular weight > 100,000, this resolution did not prevail, and no subsequent regulation has restricted use. Because of the acknowledged carcinogenic properties of degraded in animal models and the cancer-promoting effects of undegraded in experimental models, the widespread use of carrageenan in the Western diet should be reconsidered. Key words: carcinogenesis, carrageenan, carrageenase, diet, furcelleran (furcellaran), hydrolysis, inflammatory bowel disease, nutrition, poligeenan, promoter, sulfated polysaccharide. Environ Health Perspect 109:983-994 (2001). [Online 24 September 2001] http://ehpnet1.niehs.gov/docs/2001/109p983-994tobacman/abstract.html

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.

Conclusion

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.


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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.
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