The use of metabolising systems for in vitro testing of endocrine disruptors.

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine (BrdU) incorporation.

Allergic contact dermatitis is a serious health problem. Over the last decade, the murine local lymph node assay (LLNA) has been developed to detect chemical allergens, and international validation studies have been conducted. We have tried to establish an alternative non-radioisotopic endpoint for the LLNA by using 5-bromo-2'-deoxyuridine (BrdU) incorporation in place of radioisotopes, such as [3H]thymidine, employed in the standard method. BrdU was given as a single administration at 5 mg/animal 2 days following three consecutive daily applications of a test chemical. BrdU incorporation into draining lymph node cells was measured using an enzyme immunosorbent assay technique. In this study, p-benzoquinone(PBQ), trimellitic anhydride (TMA), citral(CT) and dextran (DEX) were used as pilot chemicals. PBQ, TMA and CT, which are classified as moderate to strong sensitizers in the guinea pig maximization test and were positive in the original LLNA, were also found to elicit positive responses in the alternative LLNA using BrdU incorporation. In contrast, DEX tested negative in the modified assay consistent with previous guinea pig and LLNA data. Consequently, the modified LLNA endpoint using BrdU incorporation may represent a useful alternative to the standard assay in situations, where there is a need to avoid the use of radioisotopes.

Hepatic alpha(2u)-globulin mRNA levels and diethylstilbestrol-associated testicular atrophy in rats.

Biosynthesis of alpha(2u)-Globulin (alpha(2u)-g) is under multihormonal regulation. In this study, we investigated histopathologic changes in the testis and hepatic alpha(2u)-g messenger ribonucleic acid (mRNA) levels in male rats after administration of the potent estrogen diethylstilbestrol (DES) at 0. 01, 0.1, or 1 mg/kg/day by gavage for 14 days. DES treatment decreased hepatic alpha(2u)-g mRNA levels in a dose-dependent manner accompanied by atrophic histopathologic changes in the testis. In addition, alpha(2u)-g mRNA levels were lowest in animals with the most marked testicular changes. Hepatic alpha(2u)-g mRNA may be a useful biomarker for the evaluation of endocrine disruption in male rats.

Streptavidin-reactive protein in Fasciola hepatica.

Streptavidin-binding protein was found in an extract of Fasciola hepatica, and the protein was analyzed by the inhibition assay using avidin or D-biotin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using peroxidase-conjugated streptavidin. Inhibition assays revealed that the protein could bind to streptavidin by a mechanism similar to the avidin-biotin reaction, and the molecular size of the protein was estimated by SDS-PAGE to be 71.7 kDa.

Inter-laboratory validation study of the Skin2 Dermal Model ZK1100 and MTT cytotoxicity assay kits.

An inter-laboratory validation study was conducted to evaluate the potential of 4 chemicals to cause irritation with utilizing the Skin2 Dermal Model ZK1100 kit developed by Advanced Tissue Sciences, Inc. (formerly Marrow-Tech, Inc., La Jolla, California, USA). The chemicals tested were sodium dodecyl sulfate (SDS), 1-n-hexadecyl-pyridinium chloride monohydrate (CC), ethanol (EtOH), and dimethyl sulfoxide (DMSO). Eleven Japanese institutions participated in this validation research to evaluate the usefulness of the Skin2 Model ZK1100 kit in accordance with an identical protocol. None of the participating laboratories had previously used the Skin2 Model ZK1100 kit. The MTT-50 value obtained in the individual institutions was 42 to 91 micrograms/ml for SDS, 2.7 to 8.6 micrograms/ml for CC, 2.0 to 9.3% for EtOH, and 11.5 to 21.9% for DMSO. Reproducibility was reasonably good as noted when one test chemical was repetitively tested by the same investigator. MTT-50 values obtained with the present method correlated with DS20 values obtained with Draize's method (r = 0.9881) in one of the participant institutions. The irritation study using the Skin2 Model ZK1100 kit was easy to perform and generated quantitative data. When the test was repeated, reproducibility was demonstrated with a variation of less than 2 sigma. These data suggested that this newly developed in vitro method would be useful in toxicity screening studies in terms of both time and cost, and would serve as a useful alternative to the conventional methods of the eye irritation study.

H-2 haplotype and sex-related differences in IgG response to ovalbumin in mice.

Specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in 7 strains of male and female mice after immunization with ovalbumin. Also, H-2 haplotype and sex-related differences in IgG response to ovalbumin were evaluated using statistical methods, slope ratio assay and parallel line assay. H-2k strain mice (C3H/HeN and CBA/JN) showed higher IgG responsiveness to ovalbumin than H-2d (BALB/cAnN and DBA/2 N) and H-2b (C57BL/6 N) mice. With regard to the sex-related differences in IgG response to ovalbumin, females in some strains showed higher IgG response than males, but some strains showed no sex-related differences, and sex-related differences in IgG response to ovalbumin did not relate to their H-2 haplotypes. These results may be caused by other immune response genes which control the sex-related immune response than H-2 or other unknown factors.

The relationship between the hair cycle and the age or daily body weight gain in rabbits.

The relationship between the hair cycle and the age or daily body weight gain (daily gain) in rabbits was investigated. A survey of the ages of rabbits used for the dermal toxicological studies in our laboratories over the last 4 years was done, followed by an experiment to confirm the results of the survey. Although there seemed to be no relationship between the hair cycle and the daily gain, the results showed that the hair cycle was closely related to the age of the animal, and the animals in the telogen stage may be obtained efficiently at about 17 wks of age.

Cloning and sequence of guinea pig interleukin 2 (IL-2).

Interleukin 2 (IL-2) is a T-cell proliferation factor released from TH0- and TH1-type helper T-cells and is an essential cytokine for certain immune responses. We reported here cloning and sequence of IL-2 cDNA in guinea pigs, which have been used for a long time in various immunological experiments and in vivo screening tests for skin sensitization potential of chemicals. Consequently, a cDNA clone was obtained encoding guinea pig IL-2 of 520 bp in length, which contained a complete open reading frame. Alignment of the amino acid sequence with human IL-2 indicates that guinea pig IL-2 is composed of 20 amino acids (aa) of a signal peptide and 132 aa of a mature peptide with a predicted molecular weight of 15133. Guinea pig IL-2 has an amino acid homology of 62% with human IL-2, 52% with murine IL-2, and 55% with rat IL-2. In addition, guinea pig IL-2 has a possible N-linked glycosylation site as seen in bovine and porcine IL-2.

Changes in serum alpha2u-globulin levels in male rats given diethylstilbestrol and applicability to a screening test for endocrine-disrupting chemicals.

alpha2u-Globulin (AUG) is a major rat urinary protein, which has a molecular weight of 16 kDa (kidney type) or 19 kDa (native type). The biosynthesis of this protein is under multi-hormonal regulation. In this study, we investigated changes in serum AUG level and their association with changes in the reproductive organs of male rats after the administration of the estrogenic chemical, diethylstilbestrol (DES) at doses ranging from 0.01 mg/kg per day to 100 mg/kg per day by gavage for 14 days. Our aim was to establish basic data for the development of a new screening method for endocrine disrupting chemicals based on serum AUG levels. DES treatment decreased the weight of testes in a dose-dependent manner; and was accompanied by atrophic histopathological changes in testes. Testis weights were significantly decreased by the group given 1 mg/kg per day DES; however, histopathological abnormalities were found in the group given 0.1 mg/kg per day DES. In four of five animals in the group given 1 mg/kg per day there was no significant decrease in testis weight and only a slight or moderate degeneration of the pachytene spermatocytes. Despite these findings, serum AUG levels in this group decreased markedly, while the serum AUG level markedly decreased even in the animals with no histopathological change in the 1 mg/kg per day or 0.1 mg/kg per day groups with no histopathological change also showed decreased serum AUG level. These results suggest that the serum AUG level may be a sensitive parameter for detecting the activity of estrogenic chemicals in intact male rats. Although a uterotropic assay has been proposed for immature female or ovariectomized female rats and is currently undergoing validation studies internationally, there is no screening method for estrogenic chemicals in intact male animals. More data on AUG changes by treatment with other estrogenic chemicals are needed in order to determine the sensitivity and specificity of this response to estrogens. Nonetheless, an AUG-based screening test for estrogenic chemicals may be useful owing to its applicability to conventional toxicity studies and an apparently higher sensitivity of this parameter compared to organ weight change or histology of testis in intact male rats and applicability to conventional toxicity studies.

IgE responses to Fasciola hepatica extract in several inbred mouse strains.

IgE responses to Fasciola hepatica extract in 6 strains of mice (C3H/HeN, CBA/JN, DBA/2N, BALB/cAnN, C57BL/6N and NZBWF1) were investigated. Serum anti-F. hepatica IgE antibodies were determined by passive cutaneous anaphylaxis (PCA). DBA/2N and BALB/cAnN mice showed higher IgE responses than other strains.