Fundamental and Applied Toxicology最新文献

A guinea pig intratracheal test was used to set occupational operating guidelines for new enzyme proteins used in the detergent industry. In these studies, animals were intratracheally dosed with different levels of enzyme protein and sera from the animals were titered for allergic antibody to the enzyme. The amount of antibody produced to an enzyme was compared to the amount of antibody produced to the same protein doses of Alcalase, for which effective operating guidelines exist. These comparisons were used to determine if a new enzyme was more potent, less potent, or equivalent to Alcalase; operating guidelines were then established for the new enzyme. Termamyl was about 10-fold more potent than Alcalase and the protease subtilisin B was shown to be less potent. Another protease, Savinase, was shown to be equivalent in potency to Alcalase. The operating guidelines for Termamyl were adjusted lower, whereas the operating guidelines for the proteases were set the same as that of Alcalase. Under these conditions, we would predict that sensitizations to new enzymes would be comparable to or lower than the sensitizations to Alcalase. Prospective evaluation of skin prick test data of factory workers showed that sensitizations to Termamyl and Savinase were similar to sensitizations to Alcalase. The sensitizations to subtilisin B were lower than those to Alcalase. During this time period (7 years), only three respiratory incidents (rhinitis) were reported, demonstrating that employees with positive skin prick tests can continue to work. These comparisons indicate that the guinea pig intratracheal test is a good animal model for evaluating enzymes as respiratory allergens and that the data generated can be used to set operating guidelines for occupational allergens.

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market.In vitroindicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate inSalmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r= 0.7662 for cytotoxicity andr= 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

The effects of 1,4-dichlorobenzene (DCB) have been compared in male F344 rats given 0 (corn oil control), 25, 75, 150, and 300 mg/kg DCB and male B6C3F₁mice given 0 (corn oil control), 300, and 600 mg/kg DCB by daily oral gavage five days per week for 1, 4, and 13 weeks. The two highest rat and both mouse dose levels were the same as those employed in a NTP bioassay, where DCB produced kidney tumors in male rats and liver tumors in mice. DCB produced significant dose-related increases in relative liver weight in both the rat and the mouse which was associated with, respectively, mild and marked centrilobular hypertrophy. Administration of DCB also produced a sustained induction of microsomal cytochrome P450 content and 7-pentoxyresorufinO-depentylase activity in both species. Western immunoblotting studies demonstrated that DCB induced CYP2B isoenzyme(s) in both rat and mouse liver microsomes. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2′-deoxyuridine in study Weeks 0–1, 3–4, and 12–13. In the rat hepatocyte labeling index values were only increased in animals given 300 mg/kg DCB for 1 week, whereas hepatocyte labeling index values were significantly increased in mice given 300 and 600 mg/kg DCB for 1 and 4 weeks. DCB treatment produced significant increases in rat renal P₁/P₂proximal tubule cell labeling index values at all time points, whereas little effect was observed in mouse kidney. The observed species difference in DCB-induced liver tumor formation may reflect the greater sensitivity of the mouse to tumor promotion by a CYP2B inducer. For the kidney, the present data provides further evidence that while DCB-induced α_2U-globulin nephropathy is associated with a sustained stimulation of cell replication in male rat renal proximal tubule cells, this effect is not observed in the male mouse.

The effects of fumonisin B₁(FB₁) on the immune system of Sprague–Dawley rats were investigated. Groups of male and female rats (10 rats/group) were gavaged daily for 14 days with doses of 0, 5, 15, and 25 mg/kg body wt/day and the primary (IgM) response to sheep red blood cells expressed as plaque-forming cell numbers/10⁶spleen mononuclear leukocytes (PFC/10⁶splenocytes) and PFC/spleen was determined. There was a significant dose-related linear trend toward decreased PFC/10⁶splenocytes (p= 0.003) and PFC/spleen cells (p= 0.001) in the male rats. Body weights, expressed as a percentage of the control, were significantly reduced (p= 0.002) in the male rats administered 15 and 25 mg/kg doses. The PFC numbers in female rats were not affected significantly by treatment (p> 0.05). For the remaining immunotoxicity studies, groups of male rats (10 rats/group) were gavaged with FB₁doses of 0, 1, 5, and 15 mg/kg body wt/day for 14 days. There was a weakly significant dose-related trend toward increased numbers of serum immunoglobulin class G (p= 0.04). Also a significant dose-related increase (p= 0.013) inListeria monocytogenesnumbers was observed in the spleen at 24 hr postinfection. Treatment did not have a significant effect on organ weights, hematology, mitogen-induced lymphocyte transformation, calcium mobilization, the numbers of leukocytes and T-lymphocyte subsets, the natural killer cell activity, and phagocytosis (p≥ 0.05). These observations suggested that FB₁may have indirect consequences for human health and warrant further investigations.

Effects of Polychlorinated Biphenyls (PCBs) on Brain Tyrosine Hydroxylase Activity and Dopamine Synthesis in Rats. Choksi, N. Y., Kodavanti, P. R. S., Tilson, H. A., and Booth, R. G. (1997).Fundam. Appl. Toxicol.39, 76–80.

Literature reports suggest that polychlorinated biphenyls (PCBs) may alter dopaminergic neurotransmission in mammalian forebrain.In vitro,PCBs can decrease dopamine levels in PC 12 cells and studies of the structure–activity relationship (SAR) indicate thatortho-substituted (non-coplanar) PCB congeners are more active thanpara-substituted (coplanar) congeners. This report tested the hypothesis thatortho-substituted PCBs can selectively (vspara-substituted congeners) decrease dopamine synthesis in mammalian forebrain by inhibiting the activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis.In vitroeffects of individual PCB congeners on activity of striatal tyrosine hydroxylase from two different rat strains were assessed. It was found that certainortho-substituted PCB congeners (e.g., 2,2′-DCB) can inhibit tyrosine hydroxylase activity and dopamine synthesis by nearly 40% in minces of corpus striatum prepared from Sprague–Dawley and Long–Evans hooded rats. Comparatively, theortho,meta-substituted PCB congener 2,2′,5,5′-TeCB inhibited tyrosine hydroxylase activity only in striatal minces obtained from Sprague–Dawley rats, suggesting that genetic factors may influence the susceptibility of mammals to effects of PCBs that compromise brain dopamine synthesis. The PCB-induced inhibition of tyrosine hydroxylase activity in mammalian forebrain observed here appears to occur through indirect and as yet unknown mechanisms.

Ovaries from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) bioassays were used to directly compare differential ovarian follicle counts and reproductive performance for 15 chemicals. Ovaries of 10 animals per group from 16 studies in CD-1 mice and 1 study each in C3H and C57BL/6 mice were sectioned serially at 6 μm. Counts of small, growing, and antral follicles were obtained in every 10th section. For all follicle types, younger mice had more follicles than older mice, and CD-1 mice had more follicles than age-matched animals from either inbred strain. The in-life portion of the RACB protocols demonstrated that 9 of 15 chemicals altered reproductive outcome in one or both sexes of mice, with six agents affecting females (R. E. Morrisseyet al.,1989,Fundam. Appl. Toxicol.13,747–777). Three of six female toxicants [2,2-bis(bromoethyl)-1,3-propanediol, BPD; ethylene glycol monomethyl ether, EGME; methoxyacetic acid, MAA] significantly decreased counts of small and/or growing follicles by 33 to 92% in CD-1 mice; EGME also reduced follicle counts in the other strains. Follicle counts were decreased in progeny of animals treated with EGME or its active metabolite, MAA. For BPD, reductions in follicle numbers were proportional to dose. In CD-1 mice, female toxicants di-N-hexyl phthalate, propantheline bromide, and tricresyl phosphate reduced reproductive performance but not follicle numbers. Counts were not affected by toxicants for which the susceptible sex could not be determined (bisphenol A, ethylene glycol, oxalic acid). Altered follicle counts without apparent reproductive impairment occurred in CD-1 mice at lower doses of BPD but were not observed for nontoxic chemicals. These data suggest that differential follicle counts (1) are a quantifiable endpoint of ovarian injury in conventional bioassays, and (2) in some instances, may provide a more sensitive indicator of female reproductive toxicity than fertility.

A panel of HepG2-derived cell lines (CAT-Tox [L] assay, Xenometrix), harboring stress genes consisting of a sequence for chloramphenicol acetyltransferase (CAT) under the transcriptional regulation from mammalian promoters and response elements, was exposed for 18–24 hr to aqueous suspensions of urban dusts (SRM-1648, SRM-1649, EHC-93) or PM2.5 particles (particulate matter < 2.5 μm). Expression of CAT protein was measured by enzyme-linked immunosorbent assay. Induction of the CAT genes was verified with benzo[a]pyrene (CYP1A1, cytochrome P450 1A1 promoter; GSTYa, glutathione transferase subunit Ya promoter; XRE, xenobiotic response element), cadmium sulfate, and copper sulfate (HMTIIa, metallothionein IIa promoter; HSP70, heat shock protein 70 promoter). The urban dust suspensions were active on CYP1A1, GSTYa, and XRE cell lines. SRM-1648 and SRM-1649 were twice as potent as EHC-93 per unit mass in inducing the xenobiotic-dependent responses, which correlated with contents in polycyclic aromatic hydrocarbons. These three reference particles, as well as six PM2.5 preparations collected on hi-vol filters in the Great Lakes basin, were also found to induce HMTIIa and HSP70, the magnitude of the responses correlating closely with the amount of soluble copper in the particulate preparations. The results indicate that bioavailable chemical species in the unfractionated particles can directly and quantitatively induce xenobiotic, metal, and stress-dependent responses in a target cell model, resulting in patterns of gene induction consistent with the chemical compositions of the environmental materials. We propose that cell culture models could be helpful for toxicodynamic inferences in adjunct to environmental monitoring and exposure assessments.

The effects of cinnamyl anthranilate (CA) have been compared in female B6C3F₁mice and female F344 rats fed diets containing 0–3.0% CA for periods of 1, 4, and 13 weeks. In the mouse, treatment with CA at all time points produced a marked dose-dependent increase in relative liver weight and hepatic peroxisome proliferation as demonstrated by the induction of peroxisomal (cyanide-insensitive palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidizing enzyme activities. CA produced only small increases in relative liver weight and palmitoyl-CoA oxidation in the rat and did not induce lauric acid 12-hydroxylase activity. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2′-deoxyuridine during Study Weeks 0–1, 3–4, and 12–13. After 1 week of CA treatment, labeling index values were increased in rat and to a greater extent in mouse hepatocytes. While CA treatment for 4 and 13 weeks did not increase hepatocyte-labeling index values in the rat, a sustained stimulation of replicative DNA synthesis was observed at some dietary levels in the mouse. These results demonstrate a marked species difference between the hepatic effects of CA in female B6C3F₁mice and female F344 rats. While CA is a potent peroxisome proliferator in the mouse, it is only a very weak agent in the rat. The formation of liver tumors in long-term studies, at high doses of CA, appears to be attributable to a sustained stimulation of both peroxisome proliferation and cell replication in mouse hepatocytes.

Perturbations of the balance between cell gain via mitosis and cell loss by apoptosis play a pivotal role in mediating and modifying the action of carcinogens and other toxicants in tissues such as liver, brain, the immune system, the gastrointestinal tract, and the reproductive organs. Apoptosis describes a highly conserved morphology associated with the death of many different cell types from diverse tissues. This symposium focused on induced changes in this critical balance as a key mechanism of action of a variety of diverse toxicants. In the colon, the “toxicology” of 5 fluorouracil (5FU) is entirely dependent on p53, since p53 knockouts lose the pathology of 5FU damage. Presumably, this is because DNA damage is not detected and there is no cell cycle arrest. In the testes, testicular germ cell survival is mediated by adjacent Sertoli cells via the Fas ligand (FasL)–Fas receptor (Fas) system. This system appears to mediate germ cell apoptosis after exposure to testicular toxicants such as the phthalate, mono(2-ethylhexyl) phthalate (MEHP). Interestingly, MEHP is a member of the peroxisome proliferator (PP) class of nongenotoxic carcinogens. PPs perturb both hepatocyte apoptosis and mitosis. This suppression of apoptosis occurs via activation of the peroxisome proliferator-activated receptor α (PPARα), providing a paradigm for the regulation of liver growth via activation of nuclear receptors. Similarly, the toxicological effects of dioxins are mediated via the Ah receptor (AHR), another ligand-activated nuclear receptor. This receptor upregulates a variety of genes (the Ah gene battery) associated with the toxicology of dioxins. Taken together, the data presented in this symposium illustrate to the toxicologist the need to quantitate and interpret modulations in apoptosis alongside more conventional assessments of S-phase. Although the toxicant may initiate cell damage, genes likeBcl-2,p53, Fas, PPARα, and AHR are final arbiters of the choice between death, survival, and proliferation.

Hydroquinone (HQ), catechol, and phenol exist in microgram quantities in cigarette tar and represent the predominant form of human exposure to benzene. Exposure of human T lymphoblasts (HTL)in vitroto 50 μmHQ or 50 μmcatechol decreased IL-2-dependent DNA synthesis and cell proliferation by >90% with no effect on cell viability. Phenol had no effect on HTL proliferation at concentrations up to 1 mm. The addition of HQ or catechol to proliferating HTL blocked³H-TdR uptake by >90% within 2 hr without significantly affecting³H-UR uptake, suggesting that both compounds inhibit a rate-limiting step in DNA synthesis. However, the effects of HQ and catechol appear to involve different mechanisms. Ferric chloride (FeCl₃) reversed the inhibitory effect of catechol, but not HQ, corresponding with the known ability of catechol to chelate iron. HQ, but not catechol, caused a decrease in transferrin receptor (TfR, CD71) expression, comparable to the level observed in IL-2-starved cells. HQ also inhibited DNA synthesis in cultures of transformed Jurkat T lymphocytes, primary and transformed fibroblasts, and mink lung epithelial cells, indicating that its antiproliferative effect was not restricted to IL-2 mediated proliferation. However, DNA synthesis by primary lymphocytes was more sensitive to HQ (IC50 = 6 μm) than that of the transformed Jurkat T cell line (IC50 = 37 μm) or primary human fibroblasts (IC50 = 45 μm), suggesting that normal lymphocytes may be particularly sensitive to HQ. The effects of HQ and catechol on DNA synthesis could be partially reversed by a combination of adenosine deoxyribose and guanosine deoxyribose, suggesting that both compounds may inhibit ribonucleotide reductase.