Initial insights regarding the role of p53 in maintaining sperm DNA integrity following treatment of mice with ethylnitrosourea or cyclophosphamide.

If p53 is essential to eliminate damaged spermatogenic cells, then mutagen exposure in the absence of p53 would increase sperm containing damaged DNA. p53 knockout (-/-, NULL) and wild-type (+/+, WT) mice (five/group) were exposed to ethylnitrosourea (ENU) or cyclophosphamide (CP). In phase I, mice were exposed by gavage to 0 or 60 mg/kg/day ENU or CP for four days and examined on test day (TD) 4, and in phase II, mice were exposed to 0, 6, 20, or 60 mg/kg/day ENU or CP for four days and evaluated on TD 36 when exposed spermatocytes matured. In phase I, mutagens were not directly cytotoxic to mature sperm. In phase II, WT mice were more sensitive to decreases in reproductive organ weights, whereas both genotypes had decreased sperm counts. Testicular histology revealed similar CP responses, but genotype-specific ENU responses (WT mice had depletion of elongating spermatids; NULL mice had late-stage spermatocyte/early stage spermatid loss). Ethylnitrosourea increased DNA strand breaks in WT mice. Thus, mice responded similarly to CP, suggesting a primarily p53-independent response, whereas the ENU response differed by zygosity, suggesting a role for p53. As DNA damage increased at higher ENU doses, compensatory repair pathways may operate in NULL mice.

Dose-response and operational thresholds/NOAELs for in vitro mutagenic effects from DNA-reactive mutagens, MMS and MNU.

The dose-response relationships for in vitro mutagenicity induced by methylmethanesulfonate (MMS) or methylnitrosourea (MNU) in L5178Y mouse lymphoma (ML) cells were examined. DNA adducts (N7-methylguanine, N7MeG and O(6)-methylguanine, O(6)MeG) were quantified as biomarkers of exposure. Both endpoints were assessed using 5replicates/dose (4-h treatment) with MMS or MNU (0.0069-50muM), or vehicle (1% DMSO). Mutant frequency (MF) (thymidine kinase (TK) locus) was determined using the soft agar cloning methodology and a 2-day expression period; in addition, microwell and Sequester-Express-Select (SES) methods were used for MMS. Isolated DNA was acid-hydrolyzed, and adducts quantified by LC/ESI-MS/MS, using authentic and internal standards. MF dose-responses were analyzed using several statistical approaches, all of which confirmed that a threshold dose-response model provided the best fit. NOAELs for MF were 10muM MMS and 0.69muM MNU, based on ANOVA and Dunnett's test (p<0.05). N7MeG adducts were present in all cell samples, including solvent-control cells, and were increased over control levels in cells treated with >/=10muM MMS or 3.45muM MNU. O(6)MeG levels were only quantifiable at >/=10muM MNU; O(6)MeG was not quantifiable in control or MMS-treated cells at current detection limits. Thus, (1) cells treated with

Background gene expression in rat kidney: influence of strain, gender, and diet.

In order to gain better insight into factors (strain, gender, and diet) influencing background variability in kidney gene expression, we examined the transcriptomes of male and female Crl:CD(SD)IGSBR (Sprague-Dawley [SD]) and CDF(Fischer 344)/CrlBR rats maintained for 19 days on three different diets (ad libitum [AL], diet restriction-75% of AL, and casein-based phytoestrogen-free diet). Kidney RNA was analyzed using Agilent Rat oligo microarrays (approximately 20,000 genes). Principal component analysis demonstrated that strain and gender have the most impact on the variability in gene expression, while diet had a lesser effect. The majority of the affected genes differed by a magnitude of four-fold or less between strains/gender, with some previously known to be sex-hormone regulated (SLC22A7 and SLC21A1). One gene of particular interest was ornithine decarboxylase, a significant marker of cell proliferation and tumor promotion, which was expressed at an 18-fold greater level in SD rats. Further analysis revealed that the difference in expression was due to the use of an alternate polyadenylation signal resulting in the production of two different sizes of transcripts. These results demonstrate that gender and strain have significant influence on gene expression which could be a confounder when comparing results, especially when it involves predictive fingerprint/patterns.

Incorporation of S-9 activation into an ER-alpha transactivation assay.

We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-alpha transactivation assay. 17beta-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded consistently in the assay with average EC(50) values of 9.6 x 10(-11) M and 1.2 x 10(-5) M, respectively. In the presence of a 0.1% S-9 fraction, the EC(50) for E2 was increased to 1.4 x 10(-9) M and that for MXC decreased to 4.9 x 10(-7) M, with both compounds demonstrating increased secondary metabolite formation as evidenced by HPLC analysis. Consistent with these data, metabolites of E2 and MXC exhibited decreased and increased potencies, respectively, in the assay system relative to the parent molecules. S-9 was compatible with the MCF-7 reporter assay and has the potential to enhance detection of proestrogenic materials.

Analysis of the interaction of phytoestrogens and synthetic chemicals: an in vitro/in vivo comparison.

In the evaluation of chemical mixture toxicity, it is desirable to develop an evaluation paradigm which incorporates some critical attributes of real world exposures, particularly low dose levels, larger numbers of chemicals, and chemicals from synthetic and natural sources. This study evaluated the impact of low level exposure to a mixture of six synthetic chemicals (SC) under conditions of co-exposure to various levels of plant-derived phytoestrogen (PE) compounds. Estrogenic activity was evaluated using an in vitro human estrogen receptor (ER) transcriptional activation assay and an in vivo immature rat uterotrophic assay. Initially, dose-response curves were characterized for each of the six SCs (methoxyclor, o,p-DDT, octylphenol, bisphenol A, beta-hexachlorocyclohexane, 2,3-bis(4-hydroxyphenyl)-propionitrile) in each of the assays. The six SCs were then combined at equipotent ratios and tested at 5-6 dose levels spanning from very low, sub-threshold levels, to a dose in which every chemical in the mixture was at its individual estrogenic response threshold. The SC mixtures also were tested in the absence or presence of 5-6 different levels of PEs, for a total of 36 (in vitro) or 25 (in vivo) treatment groups. Both in vitro and in vivo, low concentrations of the SC mixture failed to increase estrogenic responses relative to those induced by PEs alone. However, significant increases in response occurred when each chemical in the SC mixture was near or above its individual response threshold. In vitro, interactions between high-doses of SCs and PEs were greater than additive, whereas mixtures of SCs in the absence of PEs interacted in a less than additive fashion. In vivo, the SC and PE mixture responses were consistent with additivity. These data illustrate a novel approach for incorporating key attributes of real world exposures in chemical mixture toxicity assessments, and suggest that chemical mixture toxicity is likely to be of concern only when the mixture components are near or above their individual response thresholds. However, these data suggest that extrapolation from in vitro assays to in vivo mixture effects should be approached with caution.

A comparison of in vitro and in vivo EDSTAC test battery results for detecting antiandrogenic activity.

The androgen receptor (AR) transactivation, binding, and Hershberger assays are being developed for large-scale screening of chemicals for endocrine activity. The goal of this study was to evaluate the correlation between in vitro and in vivo antiandrogenicity assays using a variety of compounds (p,p'-DDE, flutamide (FLUT), spironolactone, procymidone, RU486, methoxychlor (MXC), benzo(a)pyrene (BAP), and selected metabolites). For the AR transactivation assay, AR(+) LNCaP prostate carcinoma cells were transfected with an inducible luciferase reporter construct (pGudLuc7ARE) and exposed for 24 h to test materials (< or = 10 microM) in the presence and absence of 1 nM of the AR agonist R-1881. Each of these materials, including the hydroxlated metabolites of BAP and MXC, produced significant antiandrogenic activity in vitro as evidenced by their inhibition of the response to R-1881. Similarly, in vitro AR binding experiments using the recombinant ligand-binding domain (LBD) of the human AR and fluorescence polarization (FP) methodology yielded IC50s comparable to that of testosterone for RU486 and 9-OH-BAP. Other parent compounds and metabolites exhibited lesser binding affinity. In vivo antiandrogenic activity was evaluated with the Hershberger assay, wherein castrated male CD rats were dosed by gavage for 10 days with (mg/kg per day): MXC (10, 50, 100, and 200), BAP (1, 10, 50, and 100), RU486 (1, 5, 10, and 25), and FLUT (10) in the presence of 0.4 mg/kg per day (sc) of testosterone propionate (TP). Neither BAP nor MXC produced significant decreases in accessory sex tissue (AST) weights relative to TP control. However, 200 MXC resulted in a significant decrease in body weight and 100 BAP significantly increased absolute and relative liver weights. RU486 (25) produced significant decreases in ventral prostate, seminal vesicle, and Cowper's gland weights without affecting body weight. FLUT (10) decreased all AST weights measured. The antiandrogenic activities of the remaining materials (p,p'-DDE, spironolactone, and procymidone) have been demonstrated in previous Hershberger assays. These data indicate the importance of including in vivo results in assessing the endocrine activity of test materials and further stress the importance of a weight of evidence approach in assessing endocrine activity of test materials.