Immature Rat Uterotrophic Assay of Bisphenol A.We used the immature rat uterotrophic assay to determine the estrogenicity of bisphenol A Bisphenol A is a chemical compound containing two phenol functional groups. It belongs to the phenol class of aromatic organic compounds. It is widely prepared and sold and various important polymers/plastics are made from it. (BPA BPA British Paediatric Association. ). We administered BPA (in sesame oil Noun 1. sesame oil - oil obtained from sesame seeds vegetable oil, oil - any of a group of liquid edible fats that are obtained from plants benniseed, sesame seed - small oval seeds of the sesame plant ) to rats subcutaneously sub·cu·ta·ne·ous adj. Located or placed just beneath the skin: subcutaneous tissue; a subcutaneous implant. sub (sc; 0, 8, 40, and 160 mg/kg/day) or orally (0, 40, 160, and 800 mg/kg/day) for 3 days beginning on postnatal postnatal /post·na·tal/ (-na´t'l) occurring after birth, with reference to the newborn. post·na·tal adj. Of or occurring after birth, especially in the period immediately after birth. day (PND (Personal Navigation Device) A portable GPS-based navigation system that can be used when walking, hiking or in any vehicle. See GPS. ) 18; rats were sacrificed 24 hr after the last administration. Uterine uterine /uter·ine/ (u´ter-in) pertaining to the uterus. u·ter·ine adj. Of, relating to, or in the region of the uterus. wet, blotted, and relative weights increased in all groups given BPA sc. After oral administration, uterine relative weight increased in 160 and 800 mg/kg BPA groups, and wet and blotted weights increased in the 800 mg/kg BPA group. Plasma concentrations of BPA at 1 hr after the last administration were detected in all groups given BPA sc and in groups given 160 and 800 mg/kg BPA orally, with a dose-response effect. The study was then reproduced under the same conditions. After sc injections, uterine wet and blotted weights increased in the 40 and 160 mg/kg BPA groups, and relative weight increased in all groups given BPA sc. By contrast, uterine wet, blotted, and relative weights increased only in the 160 and 800 mg/kg oral BPA groups. Also, to examine time-course changes in uterine weight, we administered BPA (in sesame oil) sc from PND 18 to PND 20 for 3 days at doses of 0, 8, 40, and 160 mg/kg/day; uterine weights were then measured at 6, 12, 18, and 24 hr after the last administration. Uterine wet, blotted, and relative weights increased in all BPA groups at 6 and 24 hr and in 40 and 160 mg/kg BPA groups at 12 hr. By contrast, at 18 hr, uterine wet, blotted, and relative blotted weights increased in all BPA groups and relative wet weight increased in 40 and 160 mg/kg BPA groups. The percentage increases in uterine wet and relative weights of 40 and 160 mg/kg BPA groups at 6 hr were higher than those at 24 hr relative to the controls, but the coefficient of variation Coefficient of Variation A measure of investment risk that defines risk as the standard deviation per unit of expected return. in these weights in the group given 8 mg/kg BPA at 24 hr was smaller than that at 6 hr. These findings demonstrate BPA-induced uterotrophy in the immature uterotrophic assay in rats administered 8 mg/kg/day sc and in rats given 160 mg/kg/day orally, and suggest that the autopsy at 24 hr after the last administration is suitable. Key words: bisphenol A, endocrine, immature exposure, plasma concentration, rat, uterus. Environ Health Perspect 108:1147-1150 (2000). [Online 6 November 2000] http://ehpnet1.niehs.nih.gov/docs/2OOO/108p1147-1150yamasaki/abstract.html There is concern that certain chemicals may have the potential to disturb normal sexual differentiation sexual differentiation See Hermaphroditism, hirsutism, Müllerian ducts, Precocious puberty, Pseudoprecocious puberty, Tanner staging, Testis-determining factor, Virilization, Wolffian ducts, XXX, XXY, XXXY, XYY syndromes, Y Chromosome. and development in animals and humans (1,2). Recently, the Organisation for Economic Co-operation and Development The Organisation for Economic Co-operation and Development (OECD), (in French: Organisation de coopération et de développement économiques; OCDE) is an international organisation of thirty countries that accept the principles of representative democracy and a free market (OECD OECD: see Organization for Economic Cooperation and Development. ) proposed the immature rat uterotrophic assay as one screening method for detecting the estrogenic properties of such chemicals, in which the chemical compounds are orally or subcutaneously administered for 3 days (3). Bisphenol A (BPA) has been confirmed to have weak estrogenicity in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. that is approximately 15,000 times less potent than 17 [Beta]-estradiol and interacts with both the [Alpha]-and [Beta]-estrogen receptors (4-7). It is an important chemical used principally as a monomer monomer (mŏn`əmər): see polymer. monomer Molecule of any of a class of mostly organic compounds that can react with other molecules of the same or other compounds to form very large molecules (polymers). in the manufacture of a multitude of chemical products including epoxy resins epoxy resins, group of synthetic resins used to make plastics and adhesives. These materials are noted for their versatility, but their relatively high cost has limited their use. and polycarbonate A category of plastic materials used to make a myriad of products, including CDs and CD-ROMs. , and its output has gradually increased from year to year. Uterotrophy measured by uterotrophic assay of BPA in the immature rat was previously detected after subcutaneous subcutaneous /sub·cu·ta·ne·ous/ (sub?ku-ta´ne-us) beneath the skin. sub·cu·ta·ne·ous adj. Abbr. s.c., SQ Located, found, or placed just beneath the skin; hypodermic. (sc) or oral administration of 400 mg/kg BPA (8), whereas no uterotrophy in this assay was observed in rats orally administered 5-150 mg/kg BPA (9). However, this assay has not yet been used to determine the lowest dose of BPA that can induce a uterotrophic response. One purpose of this study was to determine the estrogenic effect of BPA at low dose levels using the immature rat uterotrophic assay. We also measured the plasma concentrations of BPA 1 hr after the last administration to examine the differences in plasma concentrations between sc and oral administration routes. Because the reproducibility of the uterotrophic assay has been questioned (10), we performed a repeat study of BPA with the immature rat uterotrophic assay. Although time-course changes in uterine weight in the immature rat uterotrophic assay have been reported after the last administration of strong estrogenic compounds such as 17[Beta]-estradiol (11), there have been no reports of weak estrogenic compounds using this assay. By comparison, the OECD proposed autopsy in the immature rat uterotrophic assay at 24 hr after the last administration. Therefore, our other purpose in this study was to examine the time-course changes of uterine weight in the immature rat uterotrophic assay of BPA. Materials and Methods Study 1 Chemicals. We purchased BPA (lot 077H0666; [is greater than] 98% pure) from Kanto Chemical Co., Tokyo, Japan, and sesame oil (lot 004RYY) from Fujimi Pharmaceutical, Co., Osaka, Japan. Animals. Pregnant Crj:CD (SD) rats (at day 14 of gestation) were purchased from Charles River Charles River River, eastern Massachusetts, U.S. The longest river wholly in the state, it flows into Boston Bay after a course of about 80 mi (130 km). Navigable for about 7 mi (11 km), its estuary separates the cities of Boston and Cambridge. Japan, Inc., Shiga, Japan. At postnatal day (PND) 4, the litters were culled to eight per dam, and dams and pups were kept in polycarbonate pens until weaning weaning, n the period of transition from breast feeding to eating solid foods. weaning the act of separating the young from the dam that it has been sucking, or receiving a milk diet provided by the dam or from artificial sources. . All rats were weaned wean tr.v. weaned, wean·ing, weans 1. To accustom (the young of a mammal) to take nourishment other than by suckling. 2. at PND 17 and then housed individually in stainless steel stainless steel: see steel. stainless steel Any of a family of alloy steels usually containing 10–30% chromium. The presence of chromium, together with low carbon content, gives remarkable resistance to corrosion and heat. wire-mesh cages during the study. The immature rats were weighed, weight-ranked, and assigned randomly to each of the treatment and control groups; each group consisted of 10 rats. Body weights and clinical signs were recorded on a daily basis throughout the study. Before weaning, rats were provided with tap water and a commercial diet (CRF-1; Oriental Yeast Co., Tokyo, Japan) ad libitum ad libitum without restraint. ad libitum feeding food available at all times with the quantity and frequency of consumption being the free choice of the animal. ; after weaning, rats received water automatically and a commercial diet (MF; Oriental Yeast Co., Tokyo, Japan) ad libitum. The animal room was maintained at a temperature of 23 [+ or -] 20 [degrees] C and a relative humidity relative humidity n. The ratio of the amount of water vapor in the air at a specific temperature to the maximum amount that the air could hold at that temperature, expressed as a percentage. of 55 [+ or -] 5%, and was artificially illuminated with fluorescent light on a 12-hr light/dark cycle (0600-1800 hr). All animals were cared for according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the principles outlined in the guide for animal experimentation prepared by the Japanese Association for Laboratory Animal Science. Study design. BPA (in sesame oil) was injected sc at 8, 40, and 160 mg/kg for 3 days beginning on PND 18, or administered orally via a stomach tube at 40, 160, and 800 mg/kg for 3 days beginning on PND 18. These doses were based on the results of preliminary studies using sc or oral doses of 40, 160, and 800 mg/kg/day. The concentration and stability of BPA were confirmed. Rats were administered 2 mL/kg body weight of sesame oil containing BPA sc or 5 mL/kg orally. A vehicle control group given only sesame oil was also established for each administration route. The animals were killed approximately 24 hr after the last administration by bleeding from the abdominal vein under deep ether ether, in chemistry ether, any of a number of organic compounds whose molecules contain two hydrocarbon groups joined by single bonds to an oxygen atom. anesthesia. After necropsy necropsy /nec·rop·sy/ (nek´rop-se) examination of a body after death; autopsy. nec·rop·sy n. See autopsy. necropsy examination of a body after death. See also autopsy. , the uteri were carefully dissected dis·sect·ed adj. 1. Botany Divided into many deep, narrow segments: dissected leaves. 2. Geology Cut by irregular valleys and hills. Adj. 1. free of adhering fat and mesentery mesentery: see peritoneum. and then weighed. Plasma concentrations of BPA were measured in four female rats from each group 1 hr after the last administration. The time sampling point was determined by the results of preliminary study: mean plasma concentration of three rats given 1,000 mg/day BPA subcutaneously or orally for 3 days was higher 1 hr after the last administration than that at 0.5, 2, 4 and 6 hr after administration. Plasma samples of each group were pretreated by solid extraction column and then measured using HPLC HPLC high-performance liquid chromatography. HPLC high performance liquid chromatography. HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed . Plasma samples (250 [micro]L) were applied to the Sep-Pak C18 Cartridge (6 cc, 1 g, part WAT wat n. A Buddhist temple in Thailand or Cambodia. [Thai, from Sanskrit v  036905; Waters,
Milford, MA, USA), washed twice with 1 mL distilled water Noun 1. distilled water - water that has been purified by distillationH2O, water - binary compound that occurs at room temperature as a clear colorless odorless tasteless liquid; freezes into ice below 0 degrees centigrade and boils above 100 degrees centigrade; and 1 mL 20% acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product. , and eluted with 500 [mirco]L 70% acetonitrile. A 20 [micro]L aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) of the effluent was analyzed using an HPLC system consisting of a pump (model L-7100; Hitachi, Tokyo, Japan), an auto sampler sampler, sample piece of needlework or embroidery, of silk, cotton, or worsted, for the preservation of some pattern or as an example of the ability of a child or a beginner. In museums and private collections there are samplers dating from as early as 1643. (model L-7200; Hitachi), a column oven (model L-7300; Hitachi), a detector (model L-7480 FL; Hitachi), and a D-7000 HPLC system manager (Hitachi). An L-column ODS (Operational Data Store) A database designed for queries on transactional data. An ODS is often an interim or staging area for a data warehouse, but differs in that its contents are updated in the course of business, whereas a data warehouse contains static data. (4.6 mm i.d. x 250mm; Chemicals Evaluation and Research Institute, Tokyo, Japan) was used for the analysis. For the mobile phase, we used 43% acetonitrile in water and a flow rate of 1 mL/min. Detection was performed at 224 nm (excitation wavelength The excitation wavelength describes the light shone on a sample to transfer energy to ("to excite") a light-reactive chemical group in any compound. Its unit is usually given in nanometers (nm). ) and 310 nm (emission wavelength). Statistical analysis. Data of body weight and uterine weight were tested by Bartlett's test Bartlett's test (Snedecor and Cochran, 1983) is used to test if k samples have equal variances. Equal variances across samples is called homoscedasticity or homogeneity of variances. for homogeneity Homogeneity The degree to which items are similar. of variance. When the homogeneity of variance (p [is less than] 0.05) was evident from Bartlett's test, we performed analysis of variance (ANOVA anova see analysis of variance. ANOVA Analysis of variance, see there ). When significant (p [is less than] 0.05) treatment differences were indicated by ANOVA, we used the Student's t-test A t test is any statistical hypothesis test in which the test statistic has a Student's t distribution if the null hypothesis is true. History The t to compared each BPA group with the control. Study 2 We performed a repeat test (study 2) approximately 1 month after study 1 using the same conditions as for study 1 except that plasma concentrations were not measured. Study 3 For study 3, we used subcutaneous injection Noun 1. subcutaneous injection - an injection under the skin injection, shot - the act of putting a liquid into the body by means of a syringe; "the nurse gave him a flu shot" as the only administration route for this study, after the results of studies 1 and 2 revealed that sensitivity to sc-administered BPA was higher than with oral administration. We used the same conditions as for study 1, except the animals were sacrificed at 6, 12, 18, and 24 hr after the last administration by bleeding from the abdominal vein under deep ether anesthesia. Results Study 1 Clinical signs, body weight, and gross findings. We detected no abnormal clinical signs, including immature vaginal opening vaginal opening n. The narrowest portion of the vaginal canal, located in the floor of the vestibule, behind the urethral orifice. or gross findings. We also found no significant differences in body weight between the controls and each BPA group (Table 1).
Table 1. Body weight of rats administered BPA for 3 days beginning on
PND 18.
Start weight Final weight
Route/study Dose (mg/kg) (g) (g)
Subcutaneous
Study 1 Vehicle control 36.8 [+ or -] 2.1 47.4 [+ or -] 2.5
8 36.3 [+ or -] 2.2 46.1 [+ or -] 2.6
40 36.7 [+ or -] 2.2 45.8 [+ or -] 2.3
160 36.6 [+ or -] 2.0 45.4 [+ or -] 2.2
Study 2 Vehicle control 36.9 [+ or -] 1.2 47.5 [+ or -] 1.9
8 37.4 [+ or -] 1.8 46.5 [+ or -] 1.8
40 37.0 [+ or -] 1.0 46.8 [+ or -] 2.3
160 37.1 [+ or -] 1.3 44.7 [+ or -] 3.3
Oral
Study 1 Vehicle control 37.1 [+ or -] 2.6 46.5 [+ or -] 6.3
40 36.5 [+ or -] 1.6 45.3 [+ or -] 2.2
160 36.1 [+ or -] 2.2 45.4 [+ or -] 2.8
800 37.1 [+ or -] 1.9 45.1 [+ or -] 4.5
Study 2 Vehicle control 36.9 [+ or -] 1.1 46.6 [+ or -] 2.3
40 37.8 [+ or -] 1.1 47.2 [+ or -] 1.8
160 36.8 [+ or -] 1.5 46.2 [+ or -] 3.7
800 37.4 [+ or -] 1.5 44.5 [+ or -] 1.9
Values shown are mean [+ or -] SD.
Organ weights. Uterine wet, blotted, and relative weights were increased in all groups given BPA sc. With oral administration, however, uterine wet and blotted weights were increased in the 800 mg/kg BPA group, whereas relative weight increased in the 160 and 800 mg/kg BPA groups (Table 2).
Table 2. Uterine weight of rats administered BPA for 3 days beginning
on PND 18.
Wet
Dose weight
Route/study (mg/kg/day) (mg)
Subcutaneous
Study 1 Vehicle control 27.1 [+ or -] 3.6
8 31.1 [+ or -] 3.6(*)
40 39.5 [+ or -] 4.6(**)
160 55.7 [+ or -] 10.5(**)
Study 2 Vehicle control 27.6 [+ or -] 2.8
8 31.0 [+ or -] 4.5
40 40.7 [+ or -] 4.2(**)
160 58.4 [+ or -] 8.5(**)
Oral
Study 1 Vehicle control 27.7 [+ or -] 5.2
40 27.1 [+ or -] 2.5
160 30.5 [+ or -] 2.5
800 40.0 [+ or -] 6.6(**)
Study 2 Vehicle control 27.7 [+ or -] 3.0
40 29.8 [+ or -] 4.8
160 31.9 [+ or -] 5.1(*)
800 41.0 [+ or -]4.0(**)
Blotted
Dose weight
Route/study (mg/kg/day) (mg)
Subcutaneous
Study 1 Vehicle control 26.5 [+ or -] 3.7
8 30.3 [+ or -] 3.5(*)
40 38.6 [+ or -] 4.4(**)
160 54.7 [+ or -] 10.3(**)
Study 2 Vehicle control 27.0 [+ or -] 2.5
8 30.4 [+ or -] 4.4
40 39.7 [+ or -] 4.0(**)
160 57.4 [+ or -] 8.0(**)
Oral
Study 1 Vehicle control 27.1 [+ or -] 5.1
40 26.4 [+ or -] 2.3
160 29.8 [+ or -] 2.6
800 39.1 [+ or -] 6.7(**)
Study 2 Vehicle control 27.1 [+ or -] 3.0
40 29.0 [+ or -] 4.8
160 31.0 [+ or -] 5.1(*)
800 39.9 [+ or -] 4.0(**)
Relative wet
Dose weight
Route/study (mg/kg/day) (mg/100g)
Subcutaneous
Study 1 Vehicle control 57.2 [+ or -] 6.4
8 67.8 [+ or -] 10.4(*)
40 86.2 [+ or -] 8.7(**)
160 122.1 [+ or -] 18.9(**)
Study 2 Vehicle control 58.4 [+ or -] 7.0
8 66.6 [+ or -] 8.3(*)
40 87.1 [+ or -] 8.2(**)
160 131.1 [+ or -]21.1(**)
Oral
Study 1 Vehicle control 59.5 -[+ or -] 7.2
40 59.9 -[+ or -] 6.0
160 67.3 [+ or -] 6.1(*)
800 89.0 [+ or -] 14.6(**)
Study 2 Vehicle control 59.5 [+ or -] 6.0
40 63.1 [+ or -] 9.0
160 69.1 [+ or -] 10.3(*)
800 92.2 [+ or -]8.3(**)
Relative blotted
Dose weight
Route/study (mg/kg/day) (mg/100g)
Subcutaneous
Study 1 Vehicle control 55.8 [+ or -] 6.7
8 66.1 [+ or -] 9.7(*)
40 84.2 [+ or -] 8.4(**)
160 119.8 [+ or -] 18.7(**)
Study 2 Vehicle control 57.1 [+ or -] 6.4
8 65.2 [+ or -] 8.2(*)
40 84.8 [+ or -] 7.6(**)
160 129.0 [+ or -] 20.0(**)
Oral
Study 1 Vehicle control 58.2 [+ or -] 7.3
40 58.4 [+ or -] 5.7
160 65.8 [+ or -] 6.0(*)
800 87.1 [+ or -] 14.4(**)
Study 2 Vehicle control 58.2 [+ or -] 5.7
40 61.3 [+ or -] 9.2
160 67.4 [+ or -] 10.2(*)
800 89.5 [+ or -] 8.1(**)
Values shown are mean [+ or -] SD.
(*) Significantly different from corresponding vehicle control at p
[is less than] 0.05. (**) Significantly different from corresponding
vehicle control at p [is less than] 0.01.
Plasma concentrations. Plasma concentrations of BPA were detected in all groups given BPA sc and in groups given 160 and 800 mg/kg BPA orally, with a dose-response effect (Table 3). Comparing plasma concentrations for the same dose between sc and oral routes, BPA values were much higher in the 160 mg/kg BPA sc group than in the group given the same dose of BPA orally.
Table 3. Plasma concentration of BPA 1 hr after
last administration.
Plasma
Dose concentration
Route (mg/kg) (ng/mL)
Subcutaneous Vehicle control ND
8 94.6 [+ or -] 58.0
40 886.3 [+ or -] 56.4
160 2948.8 [+ or -] 768.8
Oral Vehicle control ND
40 ND
160 198.8 [+ or -] 88.2
800 2879.0 [+ or -] 2328.3
ND, not detected. Values shown are mean [+ or -] SD.
Study 2 Clinical signs, body weight, and gross findings. No abnormal clinical signs were detected, and there were no significant differences in body weight between the controls and each BPA group (Table 1). Uterine weight. With sc injection, uterine wet and blotted weights increased in 40 and 160 mg/kg BPA groups, whereas relative weights increased in all groups given BPA. With oral administration, uterine wet, blotted, and relative weights increased in groups given 160 and 800 mg/kg BPA orally (Table 2). Study 3 Clinical signs, body weight, and gross findings. In study 3, we observed no abnormalities in clinical signs, including immature vaginal opening and gross findings. In contrast, there were no significant differences in body weight between the control group and each BPA group. Uterine weight. Uterine wet, blotted, and relative weights increased in all BPA groups at 6 or 24 hr after the last administration; these weights also increased in the 40 and 160 mg/kg BPA groups at 12 hr (Tables 4 and 5). At 18 hr, uterine wet, blotted, and relative blotted weights were increased in all BPA groups, whereas relative wet weight increased in 40 and 160 mg/kg BPA groups. Although the percentage increases in uterine wet and relative weights of the 40 and 160 mg/kg BPA groups were higher at 6 hr after the last administration than at 24 hr, the coefficient of variation in these weights in the 8 mg/kg BPA group was smaller at 24 hr than at 6 hr.
Table 4. Absolute uterine weights of rats 6, 12, 18, and 24 hr after
last sc administration of BPA.
Dose Wet weight
Hour (mg/kg/day) (mg)(a)
6 Vehicle control 27.8 [+ or -] 5.2
8 36.5 [+ or -] 8.5(*)
40 55.6 [+ or -] 12.9(**)
160 75.9 [+ or -] 11.1(**)
12 Vehicle control 28.9 [+ or -] 6.2
8 32.2 [+ or -] 1.8
40 50.1 [+ or -] 5.8(**)
160 63.4 [+ or -] 9.8(**)
18 Vehicle control 28.3 [+ or -] 4.7
8 33.1 [+ or -] 5.3(*)
40 41.1 [+ or -] 5.6(**)
160 52.8 [+ or -] 7.4(**)
24 Vehicle control 27.6 [+ or -] 4.2
8 36.6 [+ or -] 5.1(**)
40 40.1 [+ or -] 5.5(**)
160 53.5 [+ or -] 9.4(**)
Dose Blotted weight
Hour (mg/kg/day) (mg)(a)
6 Vehicle control 27.2 [+ or -] 5.0
8 34.9 [+ or -] 7.4(*)
40 53.4 [+ or -] 12.3(**)
160 72.5 [+ or -] 10.8(**)
12 Vehicle control 28.0 [+ or -] 6.1
8 31.1 [+ or -] 1.7
40 48.6 [+ or -] 5.3(**)
160 62.0 [+ or -] 9.5(**)
18 Vehicle control 27.3 [+ or -] 4.5
8 32.4 [+ or -] 5.4(*)
40 39.9 [+ or -] 5.4(**)
160 51.6 [+ or -] 7.0(**)
24 Vehicle control 27.0 [+ or -] 4.1
8 35.7 [+ or -] 5.2(**)
40 39.4 [+ or -] 5.1(**)
160 52.3 [+ or -] 9.5(**)
Dose Wet weight CV
Hour (mg/kg/day) (% of control) (wet weight)
6 Vehicle control 100 18.7
8 131 23.2
40 200 23.2
160 273 14.6
12 Vehicle control 100 21.4
8 111 5.5
40 173 11.6
160 219 15.4
18 Vehicle control 100 16.6
8 117 16.1
40 145 13.7
160 186 14.1
24 Vehicle control 100 15.2
8 133 14.1
40 146 13.7
160 194 17.7
CV, coefficient of variation.
(a) Values shown are mean [+ or -] SD. (*) Significantly different
from corresponding vehicle control at p [is less than] 0.05.
(**) Significantly different from corresponding vehicle control at p
[is less than] 0.01.
Table 5. Relative uterine weights of rats 6, 12, 18 and 24 hr after
last sc administration of BPA.
Dose Wet weight
Hour (mg/kg/day) (mg/100g)(a)
6 Vehicle control 66.4 [+ or -] 8.1
8 87.5 [+ or -] 18.2(**)
40 132.4 [+ or -] 23.3(**)
160 189.8 [+ or -] 25.8(**)
12 Vehicle control 66.4 [+ or -] 12.1
8 78.0 [+ or -] 7.4
40 122.2 [+ or -] 17.6(**)
160 152.2 [+ or -] 20.2(**)
18 Vehicle control 62.2 [+ or -] 10.9
8 70.7 [+ or -] 8.8
40 87.3 [+ or -] 10.8(**)
160 115.7 [+ or -] 15.8(**)
24 Vehicle control 62.0 [+ or -] 7.1
8 76.3 [+ or -] 12.2(**)
40 88.9 [+ or -] 10.5(**)
160 118.0 [+ or -] 19.4(**)
Dose Blotted weight
Hour (mg/kg/day) (mg/100g)(a)
6 Vehicle control 65.0 [+ or -] 8.0
8 82.8 [+ or -] 15.8(**)
40 127.2 [+ or -] 22.0(**)
160 181.2 [+ or -] 25.8(**)
12 Vehicle control 67.3 [+ or -] 11.4
8 75.4[+ or -] 6.7
40 118.5 [+ or -] 16.1(**)
160 148.8 [+ or -] 19.8(**)
18 Vehicle control 59.8 [+ or -] 10.5
8 69.2 [+ or -] 9.0(*)
40 84.8 [+ or -] 10.4(**)
160 113.0 [+ or -] 14.7(**)
24 Vehicle control 60.7 [+ or -] 7.2
8 74.3 [+ or -] 12.4(**)
40 87.3 [+ or -] 9.8(**)
160 115.2 [+ or -] 19.5(**)
Dose Wet weight CV
Hour (mg/kg/day) (% of control) (wet weight)
6 Vehicle control 100 12.10
8 132 20.80
40 200 17.60
160 286 13.60
12 Vehicle control 100 18.20
8 112 9.40
40 176 14.40
160 219 13.30
18 Vehicle control 100 17.50
8 114 12.50
40 140 12.40
160 186 13.70
24 Vehicle control 100 11.50
8 123 16.00
40 143 11.80
160 191 16.40
CV, coefficient of variation.
(a) Values shown are mean [+ or -] SD. (*) Significantly different
from corresponding vehicle control at p [is less than] 0.05.
(**) Significantly different from corresponding vehicle control at p
[is less than] 0.01.
Discussion In the immature rat uterotrophic assay, uterine weight has been used as a sensitive parameter for evaluating estrogenic activity (12,13). The OECD has proposed uterine weight change as an end point of estrogenic activity (3). Our results confirmed that uterine weight is a good marker for evaluating the estrogenic activity of BPA. We did not use parameters such as luminal Luminal® Phenobarbital, see there cell height and cell proliferation proliferation /pro·lif·er·a·tion/ (pro-lif?er-a´shun) the reproduction or multiplication of similar forms, especially of cells.prolif´erativeprolif´erous pro·lif·er·a·tion n. because the usefulness of these parameters is unclear. Uterotrophy caused by 400 mg/kg BPA, either sc or oral administration, has been detected by the immature rat uterotrophic assay (8), but such a change has not yet been established for low doses. The present findings demonstrate that rats given a low dose of BPA, such as 8 mg/kg, show the endocrine-disrupting effect. In studies 1 and 2, uterotrophy was estimated by both wet and relative weight changes, and reproducibility of the immature rat uterotrophic assay of BPA was confirmed. In study 2, uterine wet and blotted weights increased in groups administered 40 and 160 mg/kg BPA sc and in groups administered 160 and 800 mg/kg BPA orally, whereas in study 1 these weights increased in all sc BPA groups and in the group given 800 mg/kg BPA orally. Uterotrophy in the immature rat uterotrophic assay was usually determined by the change in wet weight because this weight was almost totally uninfluenced Adj. 1. uninfluenced - not influenced or affected; "stewed in its petty provincialism untouched by the brisk debates that stirred the old world"- V.L.Parrington; "unswayed by personal considerations" unswayed, untouched by the body weight changes (14,15). Because there were differences in uterine wet weights between studies 1 and 2, uterotrophy may be better estimated by changes in both wet weight and relative weight. That uterotrophic activity was more sensitive to sc injection of BPA than oral administration reflects the differences in kinetics kinetics: see dynamics. Kinetics (classical mechanics) That part of classical mechanics which deals with the relation between the motions of material bodies and the forces acting upon them. and bioavailability bioavailability /bio·avail·a·bil·i·ty/ (bi?o-ah-val?ah-bil´i-te) the degree to which a drug or other substance becomes available to the target tissue after administration. bi·o·a·vail·a·bil·i·ty n. between these two routes. The mean plasma concentration of BPA in the sc 160 mg/kg BPA group was much higher than in the group given the same dose orally; this reflects the difference in extent of uterotrophy resulting from the two administration routes. In this comparative study of the time-course changes in BPA-induced uterotrophy, the sensitivity to BPA as measured by both uterine wet weight and relative weight was higher at 6 or 24 hr after the last administration than at 12 and 18 hr. The percentage increases in wet weight and relative weight of 40 and 160 mg/kg BPA groups at 6 hr was higher than that at 24 hr relative to controls, but the coefficient of variation for both weights in the 8 mg/kg BPA group was lower at 24 hr than at 6 hr. We therefore suggest that autopsy at 24 hr after the last administration is more suitable, based on the coefficient of variation at low dose levels. Reel et al. (11) reported that uterine weights increased until 6 hr after 17[Beta]-estradiol administration and then decreased gradually; uterine weights also increased at 24 hr. The present results demonstrate that the OECD recommendation for autopsy at 24 hr after the last administration is reasonable for rat utertrophic assays. REFERENCES AND NOTES (1.) McLachlan JA. Functional toxicology toxicology, study of poisons, or toxins, from the standpoint of detection, isolation, identification, and determination of their effects on the human body. Toxicology may be considered the branch of pharmacology devoted to the study of the poisonous effects of drugs. : a new approach to detect functionally active xenobiotics. Environ Health Perspect 101:386-387 (1993). (2.) McLachlan JA, Korach KS. Symposium on Estrogens Estrogens Hormones produced by the ovaries, the female sex glands. Mentioned in: Acne, Polycystic Ovary Syndrome estrogens (es´trōjenz), n. in the Environment, III. Environ Health Perspect 103 (suppl 7):3-4 (1995). (3.) OECD. OECD Guidance and Protocol for the Prevalidation of the Rat Uterotrophic Screening Assay. ENV/JM/ TG/EDTA(99)2/ANN1. Paris:Organisation for Economic Co-operation and Development, 1999. (4.) Feldman D, Krishnan A. Estrogens in unexpected places: possible implications for researchers and consumers. Environ Health Perspect 103(suppl 7):129-133 (1995). (5.) Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier C J, McDonnell DP. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor Steroid hormone receptors are intracellular receptors (typically cytoplasmic) that perform signal transduction for steroid hormones. Steroid hormone receptors are part of the nuclear receptor family that include a group of homologous structured receptors (type II receptors) that gene transcription Gene transcription The process by which genetic information is copied from DNA to RNA, resulting in a specific protein formation. Mentioned in: Gene Therapy assay. Toxicol Appl Pharmacol 143:205-212 (1997). (6.) Kuiper GGJ, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of the ligand ligand (lĭg`ənd), charged or uncharged molecule with one or more unshared pairs of electrons that can attach to a central metallic atom or ion to form an aggregate known as a complex ion (see chemical bond). binding specificity and transcript tissue distribution of estrogen receptors estrogen receptor A protein of a superfamily of nuclear receptors for small hydrophilic ligands–eg, steroid hormones, thyroid hormone, vitamin D, retinoids; the presence of ERs in breast CA generally is associated with a better prognosis, as they respond to [Alpha] and [Beta]. Endocrinology 138:863-870 (1997). (7.) Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, Novillo-Fertrell A, Pedraza V, Soto AM, Sonnenschein C. Estrogenicity of resin-based composites and sealants used in dentistry dentistry, treatment and care of the teeth and associated oral structures. Dentistry is mainly concerned with tooth decay, disease of the supporting structures, such as the gums, and faulty positioning of the teeth. . Environ Health Perspect 104:298-305 (1996). (8.) Ashby J, Tinwell H. Uterotrophic activity of bisphenol A in the immature rat. Environ Health Perspect 106:719-720 (1998). (9.) Gould JC, Leonard LS, Maness SC, Wagner BL, Conner K, Zacharewski T, Safe S, McDonnell DP, Gaido KW. Bisphenol A interacts with the estrogen receptor [Alpha] in a distinct manner from estradiol estradiol /es·tra·di·ol/ (es?trah-di´ol) (es-tra´de-ol) the most potent estrogen in humans; pharmacologically, it is often used in the form of its esters (e.g., e. cypionate, e. . Mol Cell Endocrinol 142:203-214 (1998). (10.) Ashby J, Elliott BM. Reproducibility of endocrine disruption data. Regul Toxicol Pharmacol 26:94-95 (1997). (11.) Reel JR, Lamb JC, Neal BH. Survey and assessment of mammalian estrogen biological assays for hazard characterization. Fundam Appl Toxicol 34:288-305 (1996). (12). Branham WS, Zehr DR, Chen JJ, Sheehan DM. Uterine abnormalities in rats exposed neonatally to diethylstilbestrol diethylstilbestrol: see DES. , ethynylestradiol, or clomiphene citrate clomiphene citrate (klō´m  fēn´ sit´rāt),n brand names: Clomid, Serophene, Milphene; drug class: . Toxicology 51:201-212 (1988). (13.) Branham WS, Zehr DR, Sheehan DM. Differential sensitivity of rat uterine growth and epithelium hypertrophy hypertrophy (hīpûr`trəfē), enlargement of a tissue or organ of the body resulting from an increase in the size of its cells. Such growth accompanies an increase in the functioning of the tissue. to estrogens and antiestrogens. Proc Soc Exp Biol Med 203:297-303 (1993). (14.) O'Connor JC, Cook JC, Craven SC, Pelt pelt the undressed, raw skin of a wild animal with the fur in place. If from a sheep or goat there is a short growth of wool or mohair on the skin. CSV (1) (Comma Separated Value) Same as comma delimited. (2) (Computer System Validation) See software validation. CSV - comma separated values , Obourn JD. An in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body. in vi·vo adj. Within a living organism. in vivo adv. battery for identifying endocrine modulators that are estrogenic or dopamine dopamine (dōp`əmēn), one of the intermediate substances in the biosynthesis of epinephrine and norepinephrine. See catecholamine. dopamine One of the catecholamines, widely distributed in the central nervous system. regulators. Fundam Appl Toxicol 33:182-195 (1996). (15.) Odum J, Lefevre PA, Tittensor S, Paton D, Routledge EJ, Beresford NA, Sumpter JP, Ashby J. The rodent rodent, member of the mammalian order Rodentia, characterized by front teeth adapted for gnawing and cheek teeth adapted for chewing. The Rodentia is by far the largest mammalian order; nearly half of all mammal species are rodents. uterotrophic assay: critical protocol features, studies with nonyl Non´yl n. 1. (Chem.) The hydrocarbon radical,  lz),n. , and comparison with a yeast estrogenicity assay. Regul Toxicol Pharmacol 25:176-188 (1997). Address correspondence to: K. Yamasaki, Chemicals Assessment Center, Chemicals Evaluation and Research Institute, 3-822, Ishii, Hita, Oita 087-0061, Japan. Telephone: +81-973-24-7211. Fax: +81-973-23-9800. E-mail: yamasaki-kanji@ hita.cerij.or.jp This work was supported by a grant from the Ministry of International Trade and Industry The Ministry of International Trade and Industry (通商産業省 Tsūsho-sangyō-shō or MITI) was one of the most powerful agencies in the Japanese government. . Received 4 April 2000; accepted 24 July 2000. Kanji (human language, character) kanji - /kahn'jee/ (From the Japanese "kan" - the Chinese Han dynasty, and "ji" - glyph or letter of the alphabet. Not capitalised. Plural "kanji") The Japanese word for a Han character used in Japanese. Yamasaki, Masakuni Sawaki, and Mineo Takatsuki Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Oita, Japan |
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