Fundamental and Applied Toxicology最新文献

The 3-demethylation of caffeine can be used as an index of cytochrome P450 CYP1A2 activityin vivo.We compared the plasma levels of caffeine and the 3-demethylated metabolite, 1,7-dimethylxanthine, in six common inbred strains (A/J, P/J, BALB/cJ, C3H/HeJ, AKR/J, and SWR/J) and one inbred strain (APN) derived in our laboratory from outbred Swiss–Webster mice on the basis of its relative susceptibility to acetaminophen-induced hepatotoxicity. We found significant variations between a number of the common strains, all of which produced significantly higher caffeine 3-demethylation indices than our APN strain. In three of the six common strains, there was a significant difference between males and females, with the females having consistently lower 1,7-xanthine/caffeine ratios. HepaticCyp1a2expression was compared between APN and C3H/HeJ males. Microsomal methoxyresorufin O-demethylation, acetanilide 4-hydroxylation, and CYP1A2 immunoreactive protein levels were significantly higher in C3H/HeJ relative to APN mice, as were hepatic CYP1A2 mRNA levels. These results indicate the importance of strain and gender to the outcome of pharmacological or toxicological studies involving CYP1A2-mediated metabolism, as well as the suitability of the plasma 1,7-dimethylxanthine/caffeine ratio as a marker of CYP1A2 activity in the mouse. The striking differences observed between the APN and C3H/HeJ mice suggest that these strains may be suitable for a genetic analysis of the regulation of the basal expression of CYP1A2, a key enzyme in procarcinogen activation.

Tributyl phosphate (TBP) produces tumors of the bladder urothelium in rats at high doses (700 and 3000 ppm), with greater effects in males than in females. TBP does not produce tumors in mice and it is nongenotoxic. The dose response of TBP effects on urine and urothelium was evaluated in male Sprague–Dawley rats at 0, 200, 700, and 3000 ppm of the diet, 10 rats per group, for 10 weeks. Another group received 3000 ppm TBP plus 12,300 ppm NH₄Cl to evaluate the effect of urinary acidification. An additional group of 10 rats received 12,300 ppm NH₄Cl. A high-dose recovery group (10 weeks 3000 ppm TBP, then 10 weeks control diet) was included to evaluate reversibility. Urine chemistries for control and TBP-treated animals were similar except for a slight decrease in osmolality and creatinine at the highest dose. Scanning electron microscopic examination of the urine of TBP-treated rats showed no increased or abnormal crystalluria, urinary precipitate, or calculi. The urothelial effects were seen at the two highest doses, but were most severe at 3000 ppm TBP, with ulceration and hemorrhage into the bladder lumen and consequent diffuse papillary and nodular hyperplasia. Dietary NH₄Cl acidified the urine but did not prevent the urothelial toxicity and regeneration. The bladder epithelial changes were reversible, but the ulcer repair process was accompanied by submucosal fibrosis. TBP at high doses appears to produce urothelial cytotoxicity with marked regenerative hyperplasia which is reversible upon withdrawal of treatment. The cytotoxicity is likely due to the direct effect of TBP or its metabolites rather than an indirect consequence of urinary changes.

Groups of guinea pigs of the Hartley strain were sensitized to toluene diisocyanate (TDI) by combined single intradermal injection and repeated inhalation exposure (3 h/day for 5 consecutive days) to 0, 3.8, 11, 26, 46, and 51 mg TDI/m³air. One group of animals was sensitized by intradermal injection only. Sham-exposed and TDI–polyisocyanate resin-sensitized guinea pigs served as controls. Three weeks after the first encounter with the inducing agent, animals were challenged with the free TDI (approximately 0.5 mg/m³) and 1 week later with TDI–guinea pig serum albumin conjugate. Breathing patterns were analyzed by objective mathematical procedures taking into account the intensity and duration of the respiratory rate exceeding ±3 standard deviations of the individual prechallenge exposure period. In none of the animals challenged with TDI were conclusive immediate-onset respiratory responses identified. During the TDI conjugate challenge a characteristic increase in respiratory rate was observed in all groups sensitized with TDI. In each of the sham and TDI–resin control groups, 1 of 16 animals responded mildly to the conjugate challenge. With regard to analysis of the development of asthma-like dyspnea, the results obtained suggest that respiratory response can suitably be defined by objective mathematical analysis of breathing patterns. Moreover, the “duration” of response exceeding +3 × standard deviation of prechallenge baseline data appears to show less variability when compared to the “intensity” of response (area). It can be concluded that this method of evaluation of respiratory response may be useful to compare more quantitatively this type of data and serves the objective of decreasing potential interlaboratory variability.

This symposium focused on the research which documents benefit and toxicity in β-carotene supplementation. Reflecting on past and current studies, the panel of experts discussed: (1) the potential harm of a high intake of β-carotene on selected populations, (2) biochemical antioxidant/prooxidant mechanisms of β-carotene at the cellular level, (3) potential benefits of other carotenoids and antioxidants, and (4) future directions for research in β-carotene and other antioxidants.

The proposed Neurotoxicity Risk Assessment Guidelines (U.S. EPA, 1995cFed. Reg.60(192), 52032–52056) of the U.S. Environmental Protection Agency (EPA) were the subject of a workshop at the 1997 Meeting of the Society of Toxicology. The workshop considered the role of guidelines in the risk assessment process, the primary features, scientific basis, and implications of the guidelines for EPA program offices, as well as for industrial neurotoxicologists from the perspectives of both pesticides and toxic substances regulation. The U.S. National Academy of Sciences (NAS, 1983, Risk Assessment in the Federal Government: Managing the Process) established a framework for distinguishing risk management from risk assessment, the latter being the result of integrating hazard identification, hazard characterization, and exposure assessment data. The guidelines are intended to establish operating principles that will be used when examining data in a risk assessment context. The proposed neurotoxicity risk assessment guidelines provide a conceptual framework for deciding whether or not a chemically induced effect can be considered to be evidence of neurotoxicity. Topics in the proposed guidelines include structural and functional effects, dose–response and –duration considerations, and relationships between effects. Among the issues that must be considered are the multiplicity of chemical effects, the levels of biological organization in the nervous system, and the tests, measurements, and protocols used. Judgment of the adversity of an effect depends heavily on the amount and types of data available. The attribution of a chemically induced effect to an action on the nervous system depends on several factors such as the quality of the study, the nature of the outcome, dose–response and time–response relationships, and the possible involvement of nonneural factors. The guidelines will also serve as a reference for those conducting neurotoxicity testing, as well as establish a consistent approach to neurotoxicity risk assessment by regulators. Extending this approach through international harmonization would be advantageous to the development of products for a worldwide market. Thus, both risk assessors and regulated industries have a large stake in the guidelines to provide a framework that will lead to accurate risk assessment decisions.

Chemicals, by virtue of their varied interactions with biological molecules, are expected to differ in the way they may alter female reproduction. Reproductive toxicity may reflect effects either on the female germ cells or on various maternal processes such as ovulation, implantation, pregnancy, and parturition. In either case, the ultimate manifestation of chemical toxicity on female reproduction is a decrease in the number of normal young born. Very little information is available on the effects of chemicals that are nonhormonal in nature on the long-term ability of treated females to produce offspring. This report presents the results of long-term female total reproductive capacity (TRC) tests on 29 chemicals, including pharmaceuticals, pesticides, and alkylating and industrial agents. For each chemical, the minimum test involved an evaluation of the maximum tolerated dose administered as a single intraperitoneal injection. Females were single-pair mated with an untreated male for most of the female's reproductive life span (a minimum of 347 days posttreatment) and scored for the number of live births produced during this period. Confirmatory dominant lethal experiments or histological examinations for numbers of small follicles were carried out when mutagenic effects or cytotoxicity, respectively, were suspected as the basis for reduced fertility. Of the 29 chemicals studied, 17 had reproductive effects which may be grouped into one of three classes: (1) those that reduced the total number of young and litters per female, (2) those that reduced the total number of young but not of litters, and (3) those that had no significant effect on the total number of young produced but reduced the size of the first and/or second litters. The TRC provides a capacity for detecting a range of toxic insults upon female reproduction. Many of the chemicals were indeed shown to affect the reproductive performance of females through mutagenic and/or cytotoxic effects on follicles. In some cases, however, no causative mechanism could be identified for the observed reduction in reproductive performance. Nevertheless, with this report the number of chemicals tested by this TRC procedure has been quadrupled and the categories of chemicals tested have been substantially broadened.

Ketoconazole (KCZ) is an imidazole antifungal agent that also affects P450 enzymes of the mammalian steroidogenic system. Several steps in the ovarian steroidogenesis pathway are known to be inhibited by KCZ, but previous work has failed to address the ramifications of such inhibition with respect to early pregnancy. In initial studies, Holtzman rats (8–10/group) were administered 10–100 mg/kg KCZ during days 1–8 of pregnancy. On day 9, evaluations revealed a reduction at both 75 and 100 mg KCZ/kg in the number of implantation sites and serum progesterone levels as well as an increase in ovarian weight. The decidual cell response (DCR) was blocked by KCZ in parallel with decreased serum progesterone and increased ovarian weight, indicating direct interference with uterine function. KCZ had no effect when given to long-term-ovariectomized rats that were hormone supplemented to permit the DCR, indicating that the ovary was at least one site of KCZ action on early pregnancy. Measurement of ovarian progesterone productionin vitrofrom ovaries removed from rats treatedin vivowith KCZ indicated a decline in progesterone production, suggesting a direct effect of KCZ on ovarian steroidogenesis. These data demonstrate that KCZ can compromise early pregnancy and appears to do so by inhibiting progesterone synthesis in the ovary.

Our laboratory has developed a method of intratracheal inhalation whereby rats can be exposed to high aerosol concentrations, resulting in high lung particle burdens in a short time period with deposition occurring directly in the lower respiratory tract, thus avoiding many drawbacks of larger nose-only or whole body inhalation systems. In this report, we compare the response of rats exposed by intratracheal inhalation to “fine” (∼250 nm) and “ultrafine” (∼21 nm) titanium dioxide particles with rats exposed to similar doses by intratracheal instillation. Animals receiving particles through inhalation showed a decreased pulmonary response, measured by bronchoalveolar lavage parameters, in both severity and persistence, when compared with those receiving particles through instillation. These results demonstrate a difference in pulmonary response to an inhaled vs an instilled dose, which may be due to differences in dose rate, particle distribution, or altered clearance between the two methods.

Primary hepatocyte cultures prepared from male beagle dog liver were used to determine susceptibility of the canine liver to tetracycline-induced steatosis. The effects of the drug on mitochondrial lipid metabolism and intracellular triglyceride accumulation were monitored at the same time that steatosis was detected by light microscopy and quantitated using lipid-specific stains. Exposure of primary canine hepatocyte cultures to tetracycline for 24–48 h resulted in concentration-dependent, significant increases in the Oil Red O-stained lipid inclusions. Microscopic examination of the total stained areas suggested that increases over control levels were due primarily to the increase in the size of the lipid inclusions rather than in the number. Biochemical analyses for triglyceride content and histological staining with Nile red, another neutral lipid-specific dye, confirmed a specific increase in intracellular triglyceride following a 24-h exposure to noncytotoxic levels of tetracycline. β-oxidation studies based on the oxidation of [¹⁴C]palmitic acid or [¹⁴C]palmitoyl carnitine demonstrated a concentration-dependent inhibition of mitochondrial but not peroxisomal β-oxidation in hepatocytes after a 24-h exposure to tetracycline.In vitroincubation of tetracycline with mitochondria isolated from dog liver showed similar, concentration-dependent inhibition. This study clearly indicates that the canine hepatocyte is susceptible to tetracycline-induced steatosis. Triglyceride accumulation was concomitant with the inhibition of mitochondrial lipid metabolism, indicating that this is a primary mechanism leading to steatosis in dog hepatocytes following tetracycline exposure.

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX, MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.