Characterization of a naphthalene derivative inhibitor of retroviral integrases.

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.

Two-generation reproductive toxicity study of inhaled tertiary amyl methyl ether (TAME) vapor in CD rats.

Under Office of Prevention, Pesticides and Toxic Substances draft guidelines, CD weanling F0 rats (30 of each gender per group) inhaled tertiary amyl methyl ether vapor at 0, 250, 1500 or 3000 ppm 5 days a week and 6 h a day for 10 weeks, with vaginal cytology evaluated for weeks 8-10. The F0 animals then produced F1 offspring, with exposure 7 days a week from mating through to lactation. During the F1 prebreed exposure period, vaginal patency, preputial separation (PPS) and vaginal cytology were evaluated. The F1 animals were mated, with F2 anogenital distance measured on postnatal day zero. At F2 weaning 30 of each gender per group were selected for postwean retention, with no exposures, through vaginal patency and PPS. Body weights, feed consumption and clinical signs were recorded throughout the study. Adult F0 and F1 systemic toxicity was present at 1500 and 3000 ppm. Minor adult male reproductive toxicity was present at 3000 ppm. There were no adult effects on vaginal cyclicity, estrous cycle length, mating, fertility, pregnancy, gestational length or ovarian and uterine weights. There were no treatment-related gross or histopathologic findings in parental male or female systemic or reproductive organs. The F1 and F2 offspring toxicity was present at 1500 and 3000 ppm. The no-observable-adverse-effect level for adult systemic and offspring toxicity was 250 ppm and 1500 ppm for male reproductive toxicity (females at >3000 ppm).

Developmental toxicity evaluation of sodium thioglycolate administered topically to Sprague-Dawley (CD) rats and New Zealand White rabbits.

BACKGROUND: Sodium thioglycolate, which has widespread occupational and consumer exposure to women from cosmetics and hair-care products, was evaluated for developmental toxicity by topical exposure during the embryonic and fetal periods of pregnancy METHODS: Timed-mated Sprague-Dawley rats (25/group) and New Zealand White (NZW) rabbits (24/group) were exposed to sodium thioglycolate in vehicle (95% ethanol:distilled water, 1:1) by unoccluded topical application on gestational days (GD) 6-19 (rats) or 6-29 (rabbits) for 6 hr/day, at 0, 50, 100, or 200 mg/kg body weight/day (rats) and 0, 10, 15, 25, or 65 mg/kg/day (rabbits). At termination (GD 20 rats; GD 30 rabbits), fetuses were examined for external, visceral, and skeletal malformations and variations. RESULTS: In rats, maternal topical exposure to sodium thioglycolate, at 200 mg/kg/day (the highest dose tested) on GD 6-19, resulted in maternal toxicity, including reduced body weights and weight gain, increased relative water consumption and one death. Treatment-related increases in feed consumption and changes at the applicationsite occurred at all doses, in the absence of increased body weights or body weight change. Fetal body weights/litter were decreased at 200 mg/kg/day, with no other embryo/fetal toxicity and no treatment-related teratogenicity in any group. In rabbits, maternal topical exposure to sodium thioglycolate on GD 6-29 resulted in maternal dose-related toxicity at the dosing site in all groups; no maternal systemic toxicity, embryo/fetal toxicity, or treatment-related teratogenicity were observed in any group. CONCLUSIONS: A no observed adverse effect level (NOAEL) was not identified for maternal toxicity in either species with the dosages tested. The developmental toxicity NOAEL was 100 mg/kg/day (rats) and > or = 65 mg/kg/day (rabbits; the highest dose tested). The clinical relevance of theses study results is uncertain because no data were available for levels, frequency, or duration of exposures in female workers or end users.

Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.

Developmental toxicity evaluation of 1,2,3,4-butanetetracarboxylic acid in Sprague Dawley (CD) rats.

1,2,3,4-butanetetracarboxylic acid (BTCA), proposed as a formaldehyde substitute in the treatment of permanent press fabrics, was evaluated for developmental toxicity. Timed-mated CD rats (25 per group) received BTCA 250, 500, or 1000 mg/kg/day or vehicle (deionized/distilled water) by gavage on gestational days (gd) 6 through 19. Maternal feed and water consumption, body weight, and clinical signs were monitored throughout gestation. At termination (gd 20), confirmed-pregnant females (21 to 25 per group) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations. One maternal death, reduced body weight, and reduced weight gain were noted at the high dose; confirmed pregnancy rates were 84 to 100% for each group. There were no treatment-related effects on fetal growth, survival, or morphologic development. The maternal toxicity NOAEL and LOAEL are 500 and 1000 mg/kg/day, respectively. The developmental toxicity NOAEL is > or = 1000 mg/kg/day, and the LOAEL was not established in this study.

Evaluation of the developmental toxicity of isoeugenol in Sprague-Dawley (CD) rats.

Isoeugenol, used as a perfumery and flavoring agent, was evaluated for developmental toxicity. Timed-pregnant CD((R)) outbred albino Sprague-Dawley rats received isoeugenol (250, 500, or 1000 mg/kg/day) or vehicle (5 ml/kg corn oil) by gavage on gestational days (gd) 6 through 19. Maternal food and water consumption, body weight, and clinical signs were monitored at regular intervals throughout gestation. At termination (gd 20), confirmed-pregnant females (23-25 per group) were evaluated for gestational outcome. All live fetuses were weighed and examined for external malformations, and approximately 50% were evaluated for visceral or skeletal malformations. There were no treatment-related maternal deaths. Clinical signs associated with isoeugenol exposure included dose-related evidence of sedation and aversion to treatment (rooting behavior) in all isoeugenol groups, as well as an increased incidence of piloerection at >/= 500 mg/kg/day. Maternal body weight, weight gain, and gestational weight gain (corrected for gravid uterine weight) were reduced at all doses in a dose-related manner. Gravid uterine weight was significantly decreased at the mid and high doses, whereas maternal relative liver weight was increased at all three dose levels. During treatment (gd 6 to 20), maternal relative food consumption was significantly decreased at the high dose, and maternal relative water consumption was elevated in the mid- and high-dose groups. Prenatal mortality (resorption or late fetal death) was unaffected. At 1000 mg/kg/day, average fetal body weight/litter was decreased by 7% (male) or 9% (female). Incidences of fetal morphological anomalies were statistically equivalent among groups, except for an increase in the incidence of unossified sternebra(e), a skeletal variation, at the high dose. In summary, the maternal toxicity lowest observed adverse effect level (LOAEL) was 250 mg/kg/day based primarily on reduced body weight and gestational weight gain (corrected for gravid uterine weight), and the maternal toxicity no observed adverse effect level (NOAEL) was not determined in this study. The developmental toxicity LOAEL was 1000 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification. The developmental toxicity NOAEL was 500 mg/kg/day.

Evaluation of the developmental toxicity of formamide in Sprague-Dawley (CD) rats.

Timed-pregnant CD(R) outbred albino Sprague-Dawley rats received formamide (50, 100, or 200 mg/kg/day) or vehicle (5 ml/kg deionized/distilled water, po) on gestational days (gd) 6 through 19. Maternal food and water consumption (absolute and relative), body weight, and clinical signs were monitored at regular intervals throughout gestation. At termination (gd 20), confirmed-pregnant females (21-23 per group) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations and variations. There were no maternal deaths and no dose-related clinical signs. At 200 mg/kg/day, maternal body weight on gd 20, weight gain, and gravid uterine weight were significantly decreased. Maternal weight gain, corrected for gravid uterine weight, liver weight (absolute or relative), and food and water consumption (absolute or relative), were not affected. Formamide did not affect prenatal viability or incidences of fetal malformations or variations. Average fetal body weight/litter was decreased at 100 and 200 mg/kg/day. Fetal body weight was affected at lower daily doses than in previously published studies, possibly due to the longer total exposure period and/or lack of a recovery period between cessation of exposure and termination. In summary, the maternal toxicity no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day and the low observed adverse effect level (LOAEL) was 200 mg/kg/day under the conditions of this study. Similarly, the developmental toxicity NOAEL was 50 mg/kg/day and the LOAEL was 100 mg/kg/day.

Relationship between acrylamide reproductive and neurotoxicity in male rats.

To determine whether there is a relationship between the reproductive and neurotoxic effects of acrylamide monomer (AM), the first week of the study design of Sublet et al. ¿14 was duplicated: Long-Evans male rats were gavaged with AM in water, 25/group, at 0, 5, 15, 30, 45, or 60 mg/kg/day for 5 days (days 1 through 5). On Day 8, males were paired overnight with untreated virgin females (1 : 1) in proestrus/estrus. On day 9, males were evaluated for forelimb and hindlimb grip strength. Five males/group were perfusion fixed, 20/group were used for andrologic assessment, and all were necropsied. Perfusion-fixed sciatic nerves were examined histologically. Sperm-positive females were examined for preimplantation and postimplantation loss at midpregnancy. At 15 to 60 mg/kg/day, males exhibited significantly reduced weight gain, reduced mating, fertility, and pregnancy indices by trend analysis (significant at 60 mg/kg/d by pairwise comparison), and increased postimplantation loss and dominant lethal factor, F(L)%, at 45 and 60 mg/kg/day. At 60 mg/kg/day, the sperm beat cross frequency was increased, with no significant effects on epididymal sperm motility or concentration, and hindlimb grip strength was decreased, with no pathologic lesions in sciatic nerves. Therefore, epididymal sperm, mating, and neurotoxic effects were observed at AM doses that also resulted in increased postimplantation loss, possibly by different mechanisms.

The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits.

Nyotran is a liposomally encapsulated i.v. formulation of the antifungal polyene nystatin. This drug was evaluated in a series of reproductive toxicity studies, according to the guidelines outlined by the International Conference on Harmonization (ICH). A fertility and early embryonic development study (SEG I) and a prenatal and postnatal development (SEG III) study were conducted in rats, and embryo-fetal development (SEG II) studies were conducted in rats and rabbits. Nyotran was administered iv in all studies. In SEG I and SEG III, rats were administered daily doses of 0.5, 1.5, or 3.0 mg/kg Nyotran. In both studies, parental mortality and toxicity in the 3.0 mg/kg dose group necessitated the lowering of the high dose to 2.0 mg/kg/day. Parental toxicity, in the form of decreased body weights, decreased food consumption, and piloerection were also observed at the 1.5 mg/kg/day dose level in the SEG I and SEG III studies. Despite the parentally toxic doses in the SEG I study, there was no effect of Nyotran on F0 male or female fertility or early embryonic development of F1 offspring. In the SEG III study, lactational body weights of the F1 generation were decreased at all Nyotran dose levels. There was no effect on pre-wean developmental landmarks, but post-wean development was affected by Nyotran administration at all dosage levels. Preputional separation was delayed in the 1.5 and 3.0/2.0 mg/kg/day F1 offspring, auditory startle function was decreased in F1 females at all dose levels, and motor activity was decreased in male F1 offspring at all dose levels. However, there were no treatment-related effects on the subsequent mating of the F1 generation and resulting F2 offspring. In SEG II studies, rats and rabbits were also administered 0.5, 1.5, or 3.0 mg/kg/day of Nyotran during gestation. The high dose in these SEG II studies was not lowered, as the maternal animals were able to tolerate the shorter duration of dosing. Maternal effects in rabbits were observed only in the high-dose group and were limited to decreased food consumption and decreased absolute and relative liver weight. Decreased food consumption in high-dose dams and clinical weight loss in some animals at the mid- and high-dose levels evidenced maternal toxicity in rats. Nyotran did not have any effect on Caesarian section parameters in either rats or rabbits and no effect on the incidence of fetal malformations in rabbits. A statistically significant increase in mild hydrocephaly, observed in 4 rat fetuses, was seen at the highest dose level of 3.0 mg/kg/day. The biological significance and relationship to Nyotran treatment of this finding is not clear. This finding may represent a change in the background incidence or a change in the pattern of responsiveness of this strain of rat fetus to the test chemical. Toxicokinetic data were also collected in the SEG II rabbit and rat studies for comparison to human exposures. In both species, systemic exposure to the nystatin at effective antifungal concentrations was demonstrated. The systemic exposures in rats and rabbits were, however, considerably less than have been reported in humans administered clinical doses of 2 or 4 mg/kg/day Nyotran. Thus, humans tolerate higher dosages and systemic exposures of Nyotran relative to rats and rabbits and there is no margin of safety in either dosage level or systemic exposure to drug. Given this lack of a margin of safety and the effects on postnatal development in F1 rats, caution should be exercised when using this drug in females of childbearing potential.

Indoloquinoxaline compounds that selectively antagonize P-glycoprotein.

Tumor cells often develop drug resistance through overexpression of membrane transport proteins that effectively efflux anticancer agents. The pharmacologies of the two best-studied transporters, P-glycoprotein (Pgp) and MRP1, are partially overlapping but distinct. To improve the therapeutic potential of drug resistance reversing agents, we have developed a program to identify compounds with selectivity for Pgp or MRP1. Screening of a commercial library of compounds identified indoloquinoxaline compounds with transporter selectivity, and certain examples were synthesized and further evaluated. 1,4-Dibutoxy-6H-indolo[2,3-b]quinoxaline and 4,7-dibutoxy-2,3-dihydrobenzimidazole-2-spiro-3-indolin-2-one were synthesized by condensation of 3,6-dibutoxy-1,4-diaminobenzene and isatin. Neither compound was cytotoxic to MCF-7 cells, nor did either one affect the sensitivity of MCF-7/VP or HL-60/ADR cells at doses up to at least 20 microM, indicating that they do not antagonize MRP1. In contrast, each compound, at doses as low as 0.25 microM, sensitized NCI/ADR cells to vinblastine, actinomycin D, Taxol, and doxorubicin, indicating that they effectively reverse Pgp-mediated multidrug resistance (MDR). Furthermore, the compounds sensitized two additional cell lines that overexpress Pgp to this panel of anticancer drugs. However, these compounds did not affect the sensitivities of MCF-7 or T24 cells to these cytotoxic drugs, and did not alter the sensitivities of any of the tested cell lines to cisplatin or 5-fluorouracil. Both compounds enhanced the intracellular accumulation of [3H]vinblastine by NCI/ADR cells, but did not inhibit photoaffinity labeling of Pgp by [3H]azidopine at concentrations up to at least 100 microM. Therefore, these novel nontoxic indoloquinoxalines selectively sensitize Pgp-overexpressing cells to drugs that are subject to transport by this protein, without modulating the sensitivities of MRP1-overexpressing or non-Pgp cells to cytotoxic drugs. Because of this transporter selectivity, we predict that these compounds will be effective MDR modulators in vivo.

Pyrimidine dimer formation as a probe of nucleosome core and linker structure in situ.

The photoinduced dimerization of adjacent pyrimidines in DNA is influenced in predictable ways by DNA conformation. A method is described for determining patterns of pyrimidine dimer formation under conditions in which the chromatin is minimally perturbed. The relation of such patterns to the conformation of nucleosomal core DNA and linker DNA, as well as the interaction of histone H1 with nucleosomal DNA, is presented. Such data indicate that sharp bends in the path of DNA seen in crystals of isolated nucleosome core particles are also present in intact chromatin. They also indicate that most of the linker has very little curvature except for a small bend at its junction with the nucleosome core. The linker path inferred from such experiments supports models in which the chromatin fiber consists of a zigzag chain of nucleosomes.

Effects of lactational administration of acrylamide on rat dams and offspring.

We duplicated the study design of Husain et al. (Ind Health 1987; 25:19-28) to determine whether maternal exposure to acrylamide monomer (AM) resulted in offspring neurotoxicity. Wistar rat dams with litters (15/group) were gavaged with AM in saline at 0 or 25.0 mg/kg/d throughout lactation (pnd 0-21). Maternal feed and water consumption, body weights (BW), and Functional Observational Battery (FOB) were recorded. At weaning (pnd 21), maternal sciatic nerves were examined histologically. Male offspring were retained until pnd 91, with BW and grip strength evaluations. Dosed dams exhibited progressive toxicity, including mortality (two), severely reduced feed and water consumption, BW, and BW gain, and behavioral neurotoxicity (with no sciatic nerve pathology). Nursing offspring at 25.0 mg/kg/d exhibited increased mortality and reduced BW associated with little/no milk in stomachs. Postwean males at 25.0 mg/kg/d exhibited normal BW gain and increasing grip strength over time. Therefore, AM caused maternal toxicity; offspring effects during lactation were consistent with inanition from maternal toxicity. Postwean males exhibited recovery with no signs of AM-mediated toxicity. These results do not support the conclusions of Husain et al.

Two-generation reproduction study with para-tert-octylphenol in rats.

Octylphenol (OP) is a commercial intermediate used primarily for the production of octylphenol polyethoxylate surfactants. To determine potential reproductive toxicity of OP, a two-generation reproduction study was conducted according to U.S. EPA OPPTS Guideline 870.3800 (draft 1996). Additional measurements were made on retained F2 offspring. OP was administered ad libitum to five groups of rats (30/sex) at dietary concentrations of 0, 0.2, 20, 200, or 2000 ppm. The 0.2 ppm concentration was included to evaluate potential low dose effects. Effects were observed only at 2000 ppm, including decreased body weights in adults and during the latter portion of lactation in offspring and minor body weight-related delays in acquisition of vaginal opening and preputial separation. No effects on reproductive parameters, testes, prostate, or ovary weights or morphology, on sperm counts, motility, morphology, production, or on estrous cyclicity were observed. No estrogen-like effects were evident. The NOAELs for systemic and postnatal toxicity were 200 ppm and at or above 2000 ppm for reproductive toxicity. This study supports the increasing evidence that screening assays for estrogenic activity or studies with limited numbers of animals and/or unrealistic dose regimens are inappropriate for use in the assessment of human health and environmental risk. It does not support previous preliminary data on low dose effects of OP.

Evaluation of the developmental toxicity of methacrylamide and N,N'-methylenebisacrylamide in Swiss mice.

Timed-pregnant CD-1 outbred albino Swiss mice received either methacrylamide (MAC; 0, 60, 120, or 180 mg/kg/day) or N,N'-methylenebisacrylamide (BAC; 0, 3, 10, or 30 mg/kg/day) p.o. in distilled water on gestational days (GD) 6 through 17. Maternal clinical status was monitored daily. At termination (GD 17), confirmed-pregnant females (27-30 per group, MAC; 24-25 per group, BAC) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations. For MAC, no treatment-related maternal mortality was observed. Maternal body weight on GD 17, maternal weight gain during treatment and gestation, and corrected maternal weight gain were reduced at the high dose. Relative maternal food and water intake was not adversely affected; neurotoxicity was not observed. Relative maternal liver weight was increased at > or = 120 mg/kg/day; gravid uterine weight was decreased at 180 mg/kg/day. The maternal no-observed adverse effect level (NOAEL) was 60 mg/kg/day. The NOAEL for developmental toxicity was also 60 mg/kg/day. At > or = 120 mg/kg/day, mean fetal body weight was reduced. At 180 mg/kg/day, increased postimplantation death per litter was observed. Morphological development was not affected. The maternal NOAEL for BAC was 10 mg/kg/day. At 30 mg/kg/day, decreased maternal body weight on GD 17, maternal body weight change during treatment and gestation, corrected maternal body weight, and gravid uterine weight were observed. Relative maternal liver weight increased at 30 mg/kg/day. The developmental NOAEL was 3 mg/kg/day BAC. Mean fetal body weight was reduced at 30 mg/kg/day. At > or = 10 mg/kg/day, an increased incidence of fetal variations (extra rib) was observed, although fetal malformation rate was unaffected. MAC and BAC were not teratogenic to Swiss mice at the doses tested. BAC was more potent than MAC in causing adverse maternal and developmental effects.

Two-generation reproductive toxicity study of dietary tributyl phosphate in CD rats.

Tributyl phosphate (TBP) was tested for reproductive toxicity in rats. Thirty weanlings/sex (F0) were exposed to TBP in the diet ad libitum at 0, 200, 700, or 3000 ppm for 10 weeks and then randomly mated within groups for 3 weeks with continued exposure. F0 parents and 10 F1 weanlings/sex/dose were necropsied, and adult reproductive organs, urinary bladders (both sexes), kidneys (males), and livers (females) were evaluated histologically. Thirty F1 weanlings/sex/dose continued exposure for 11 weeks and were bred as described above. F1 parents and F2 weanlings, 10/sex/dose, were then necropsied as described above. Adult toxicity was observed in both sexes and generations at 700 and 3000 ppm; observations included reduced body weights, weight gain and feed consumption, urinary bladder epithelial hyperplasia (both sexes), renal pelvis epithelial hyperplasia only at 3000 ppm (male kidneys), and centrilobular hypertrophy (female livers). At 200 ppm, transient reductions in body weight were observed in F0 and F1 females, with urinary bladder epithelial hyperplasia in F0 males and females and in F1 males. There was no evidence of reproductive toxicity, of reproductive organ pathology, or of effects on gestation or lactation at any dose tested. Postnatal toxicity was evidenced by consistent reductions in F1 and F2 pup body weights at 3000 ppm and by occasional weight reductions in F2 litters at 700 ppm, and was associated with maternal toxicity observed at these doses and times. Under the conditions of this study, a NOAEL was not determined for adult toxicity; the NOAEL for reproductive toxicity was at least 3000 ppm and the NOAEL for postnatal toxicity was approximately 200 ppm.

Cellular kinetics of induction by oltipraz and its keto derivative of detoxication enzymes in human colon adenocarcinoma cells.

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] is a synthetic dithiolethione with chemopreventive activity against carcinogen-induced neoplasia of liver, lung, and colon in several animal model systems. Protection from tumor formation is associated with elevation of Phase II enzymes, including glutathione (GSH) transferase and NAD(P)H:quinone oxidoreductase (DT-diaphorase) in experimental carcinogenesis models in vivo. To investigate the time and dose relationships of the pharmacological action of oltipraz and to develop a model for its investigation, a human colon adenocarcinoma HT29 cell line was primarily used. In this cell line, oltipraz resulted in increased activity of both GSH transferase and DT-diaphorase. At the maximum effective concentration (100 microM), the elevation of GSH transferase was 3-fold and that of DT-diaphorase was 2-fold. The optimal duration of oltipraz exposure to HT29 cells was 24 h, following which the peak in enzyme activity was observed at 24 h after removal of the drug, and activity had almost returned to control levels after 72 h in drug-free media. Steady-state mRNA levels for DT-diaphorase were observed to increase during the period of drug exposure and remained elevated, even as catalytic activities declined to control levels, suggesting additional mechanisms for control of the activity of this enzyme. More prolonged drug exposure was associated with less induction of the detoxication enzymes, prompting an investigation of the possible toxicity of oltipraz to these cells. Although the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed inhibition of proliferation (IC50, 100 microM oltipraz), a clonogenic assay demonstrated no loss of clonogenicity. Oltipraz is known to be extensively metabolized in many species; two major metabolites include a 3-ketone (metabolite 2, M2) and a molecular rearrangement to a pyrrolopyrazine derivative (metabolite 3, M3), numerous conjugates of which are formed in vivo. To investigate the potential cause of the lag in response, we synthesized two major oltipraz metabolites (M2 and M3) and tested their efficacy in enzyme induction. The activity of DT-diaphorase was induced similarly by both oltipraz and M2 (2.6- versus 2.8-fold baseline) at 100 microM, whereas M3 was inactive at all concentrations. M2 also resulted in a 5.8-fold elevation of steady-state DT-diaphorase mRNA levels. Both enzyme activity and steady-state mRNA peaked at 24 h as with the parent compound. Thus, the oxidative desulfuration of oltipraz results in the formation of an active metabolite, but this process is not rate limiting for the induction of detoxicating enzymes. These data support the use of intermittent schedules in oltipraz in clinical trials of chemoprevention because of evidence of attenuation of response. The metabolite M2, but not M3, is as active as the parent compound and may be considered for clinical development in its own right.

The developmental toxicity of boric acid in rabbits.

Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce reproductive and developmental toxicity in laboratory rodents. In this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers of uterine implantations, resorptions, dead fetuses, and live fetuses were determined. Fetuses were weighed, and live fetuses examined for external, visceral, and skeletal defects. Maternal food intake decreased during treatment at 250 mg/kg/day and increased at >/=125 mg/kg/day after treatment. Maternal body weight (GD 9-30), weight gain during treatment, gravid uterine weight, and number of ovarian corpora lutea decreased at 250 mg/kg/day. In contrast, maternal corrected gestational weight gain increased at >/=125 mg/kg/day. Maternal liver weight was not affected. Relative (but not absolute) maternal kidney weight increased at 250 mg/kg/day, and microscopic evaluation revealed no treatment-related renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion of pregnant females with no live fetuses was increased (73% vs 0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As a result, there were only 14 live fetuses (6 live litters) available for evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily due to cardiovascular defects in 72% of high-dose fetuses vs 3% of controls. The most prevalent cardiovascular malformation (interventricular septal defect) was observed in 57% of high-dose fetuses compared to 0.6% among controls. At 250 mg/kg/day, average fetal body weight/litter was 92% of the average control weight (not statistically significant). In summary, no definitive maternal or developmental toxicity was observed at 62.5 or 125 mg/kg/day BA. Mild maternal effects and severe developmental toxicity were observed at 250 mg/kg/day.

Evaluation of the developmental toxicity of methacrylonitrile in Sprague-Dawley rats and New Zealand white rabbits.

Timed-pregnant Sprague-Dawley (CD) outbred rats and New Zealand White rabbits were dosed by gavage with methacrylonitrile (MACR) in distilled water during major organogenesis. Rats were dosed on Gestational Days (GD) 6 through 15 (0, 5, 25, or 50 mg MACR/kg/day) and rabbits on GD 6 through 19 (0, 1, 3, or 5 mg MACR/kg/day). Maternal clinical status was monitored daily during treatment. At termination (GD 20, rats; GD 30, rabbits), confirmed-pregnant females (25-26 per group, rats; 17-22 per group, rabbits) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In rats, no treatment-related maternal clinical signs or mortality were observed, nor was there any adverse effect on maternal body weight or food or water consumption. At necropsy, absolute, relative, and adjusted maternal liver weight was increased at the mid- and high-dose groups, an effect that may be indicative of induction of hepatic enzymes rather than toxicity. In the absence of any indication of maternal toxicity, the no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was >/=50 mg MACR/kg/day. The NOAEL for developmental toxicity in rats was also >/=50 mg MACR/kg/day. There was no effect of treatment on postimplantation loss, mean fetal body weight per litter, or morphological development. In rabbits, maternal mortality and clinical signs were not dose related. Maternal food consumption, body weight, and liver weight were not adversely affected by treatment. Thus, the maternal NOAEL was >/=5 mg MACR/kg/day. Maternal toxicity, including death, was observed >/=7.5 mg/kg/day in preliminary studies. The developmental NOAEL was also >/=5 mg MACR/kg/day. There was no adverse effect of treatment on postimplantation loss or fetal body weight. A significant decrease in the percentage male fetuses per litter was observed, although there was no effect on total live litter size, suggesting that the reduction in the ratio of live male fetuses in the high-dose group was not biologically significant. MACR had no adverse effect on morphological development. In summary, oral administration of MACR to rats and rabbits during organogenesis, at doses that did not cause persistent maternal toxicity (50 mg MACR/kg/day, rats; 5 mg MACR/kg/day, rabbits), also did not cause any adverse developmental effects.

Developmental toxicity NOAEL and postnatal recovery in rats fed boric acid during gestation.

Boric acid (BA), an essential plant micronutrient, occurs naturally in fruits, vegetables, and other foods. It is widely used in the manufacture of glass, ceramics, and other products. In a prior study, gestational exposure to BA was associated with developmental toxicity in the rat, including fetal growth retardation and altered skeletal morphology. In order to establish the developmental toxicity no-observed-adverse-effect level (NOAEL) in the rat, BA (0, 0.025, 0.05, 0.075, 0.1, or 0.2% in feed) was administered to timed-mated rats (60/group) from gestational day (gd) 0 to gd 20. Approximately half the dams were terminated on gd 20, and the remaining dams delivered their litters. Pup growth and viability were monitored until postnatal day (pnd) 21. Dams sacrificed on gd 20 (pnd 21) ingested average doses of 0(0), 19(19), 36(37), 55(56), 76(74), or 143(145) mg BA/kg/day. Maternal clinical signs, body weight, and food and water intake were measured at regular intervals during gestation and lactation. At termination, maternal liver and right kidney were weighed, and live fetuses (gd 20) and pups (pnd 21) were weighed, sexed, and examined for morphological anomalies (external, visceral, skeletal). Maternal effects were limited to increased relative kidney weight at 0.2% BA. Viability of the offspring was unaffected. On gd 20, fetal body weight was 94 and 88% of controls at 0.1 and 0.2% BA, but recovery was complete at birth (approximately gd 22). The incidence of short rib XIII was increased on gd 20 at > or = 0.1% BA, but only at 0.2% on pnd 21. The incidence of wavy rib was increased on gd 20 at > or = 0.1% BA, but the reversibility of this effect was confirmed on pnd 21. A slight decrease in extra lumbar ribs was observed at 0.2% BA on gd 20, and extra lumbar ribs were not found in any pups on pnd 21. Thus, the developmental toxicity NOAEL in the rat was 0.075% BA (55 mg/kg/day) on gd 20 and 0.1% BA (74 mg/kg/day) on pnd 21.

Reproductive toxicity evaluation of methylethyl ketoxime by gavage in CD rats.

Methylethyl ketoxime (CAS No. 96-29-7; MEKO; 2-butanone oxime), an antioxidant agent used in paints, resins, and adhesives, was tested for reproductive toxicity in a two-generation study with CD (Sprague-Dawley) rats. Thirty-eight-week-old rats/sex/group (F0) were administered MEKO in water, by gavage, at 0, 10, 100, or 200 mg/kg/day (at a dosing volume of 2 ml/kg), 5 days/week for 10 weeks with vaginal cytology evaluation (VCE) of F0 females during the last 3 weeks of the prebreed period. Animals were mated within groups for 3 weeks with dosing during mating, gestation, and lactation for 7 days/week. F0 parents and F1 weanlings, 10/sex/dose, were necropsied (after a 2-week postwean VCE in F0 females) with hematologic evaluation (including methemoglobin) and histology of adult livers, spleens, and reproductive organs. F1 weanlings, 30/sex/dose, were dosed for 11 weeks and mated as described above. Because of poor reproductive performance, not treatment related, F1 animals with no F2a litters were rebred to produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose, were necropsied and evaluated as described above. Inguinal mammary glands were examined histologically from all nonselected F1 and F2 (a and b) female weanlings. Adult toxicity was observed in both generations and both sexes at all doses. Treatment-related parental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day, parents exhibited dose-related reduced body weights and weight gains, reduced feed consumption, clinical signs of toxicity, and anemia with concomitant extramedullary hematopoiesis and hemosiderosis in livers and spleens (and increased spleen weights). At 10 mg/kg/day, only adult liver and spleen histologic effects were present. There was no evidence of reproductive organ or mammary glad pathology or of reproductive or postnatal toxicity at any dose tested. There was no adult "no observable adverse effect level" (NOAEL) established; the NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg/day for rats in this study.