Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles.

A multispecies, subchronic, inhalation study comparing pulmonary responses to ultrafine titanium dioxide (uf-TiO(2)) was performed. Female rats, mice, and hamsters were exposed to aerosol concentrations of 0.5, 2.0, or 10 mg/m(3) uf-TiO(2) particles for 6 h/day, 5 days/week, for 13 weeks. Following the exposure period, animals were held for recovery periods of 4, 13, 26, or 52 weeks (49 weeks for the uf-TiO(2)-exposed hamsters) and, at each time point, uf-TiO(2) burdens in the lung and lymph nodes and selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters, including inflammation, cytotoxicity, lung cell proliferation, and histopathological alterations. Retained lung burdens increased in a dose-dependent manner in all three species and were at a maximum at the end of exposures. Mice and rats had similar retained lung burdens at the end of the exposures when expressed as mg uf-TiO(2)/mg dry lung, whereas hamsters had retained lung burdens that were significantly lower. Lung burdens in all three species decreased with time after exposure, and, at the end of the recovery period, the percentage of the lung particle burden remaining in the 10 mg/m(3) group was 57, 45, and 3% for rat, mouse, and hamster, respectively. The retardation of particle clearance from the lungs in mice and rats of the 10 mg/m(3) group indicated that pulmonary particle overload had been achieved in these animals. Pulmonary inflammation in rats and mice exposed to 10 mg/m(3) was evidenced by increased numbers of macrophages and neutrophils and increased concentrations of soluble markers in bronchoalveolar lavage fluid (BALF). The initial neutrophil response in rats was greater than in mice, whereas the relative increase of macrophages was less than in mice. The neutrophilic response of rats, but not mice, declined in a time-dependent manner correlating with declining lung burdens; however, the fraction of recovered neutrophils at 52 weeks postexposure was equivalent in the two species. Consistent increases in soluble indicators of toxicity in the BALF (LDH and protein) occurred principally in rats and mice exposed to 10 mg/m(3) and diminished with time postexposure. There were no significant changes in cellular response or with markers indicating toxicity in hamsters, reflecting the capacity of these animals to rapidly clear particles from the lung. Progressive epithelial and fibroproliferative changes were observed in rats of the 10 mg/m(3) group. These lesions consisted of foci of alveolar epithelial proliferation of metaplastic epithelial cells (so-called alveolar bronchiolization) circumscribing aggregated foci of heavily particle-laden macrophages. The observed epithelial proliferative changes were also manifested in rats as an increase in alveolar epithelial cell labeling in cell proliferation studies. Associated with these foci of epithelial proliferation were interstitial particle accumulation and alveolar septal fibrosis. These lesions became more pronounced with increasing time postexposure. Epithelial, metaplastic, and fibroproliferative changes were not noted in either mice or hamsters. In summary, there were significant species differences in the pulmonary responses to inhaled uf-TiO(2) particles. Under conditions where the lung uf-TiO(2) burdens were equivalent, rats developed a more severe inflammatory response than mice and, subsequently, developed progressive epithelial and fibroproliferative changes. Clearance of particles from the lung was markedly impaired in mice and rats exposed to 10 mg/m(3) uf-TiO(2), whereas clearance in hamsters did not appear to be affected at any of the administered doses. These data are consistent with the results of a companion study using inhaled pigmentary (fine mode) TiO(2) (Bermudez et al., 2002) and demonstrate that the pulmonary responses of rats exposed to ultrafine particulate concentrations likely to induce pulmonary overload are different from similarly exposed mice and hamsters. These differences can be explained both by pulmonary respy response and by particle dosimetry differences among these rodent species.     

Acute inflammatory responses to Stachybotrys chartarum in the lungs of infant rats: time course and possible mechanisms.

Stachybotrys chartarum has been linked to building-related respiratory problems including pulmonary hemorrhage in infants. The macrocyclic trichothecenes produced by S. chartarum have been the primary focus of many investigations. However, in addition to trichothecenes this fungus is capable of producing other secondary metabolites and a number of protein factors. This study examines the effects of intact, autoclaved, and ethanol-extracted spores on the lungs of infant rats as an approach to differentiate between secondary metabolites and protein factors. Seven-day-old infant rats were exposed intratracheally to 1 x 10(5) spores/g body weight (toxic strain JS58-17) and sacrificed at various times up to 72 h. The inflammatory response was measured by morphometric analysis of the lungs and determination of inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid. Alveolar space was greatly reduced in animals exposed to fungal spores compared to phosphate buffered saline (PBS)-treated controls. The largest effects were observed in pups treated with intact spores where alveolar space 24 h after treatment was 42.1% compared to 56.8% for autoclaved spores, 51.1% for ethanol-extracted spores, and 60.6% for PBS-treated controls. The effects of different spore preparations on inflammatory cells, cytokine, and protein concentrations in the BAL fluid can be ranked as intact > autoclaved > extracted. Tumor necrosis factor alfa (TNF-alpha), interleukin 1-beta (IL-1beta), and neutrophils were the most sensitive indicators of inflammation. The difference between autoclaved (100% trichothecene toxicity, denatured/enzymatically inactive proteins) and intact (100% trichothecene activity, unaltered/released proteins) spores indicates the involvement of fungal proteins in the inflammatory response to S. chartarum and sheds new light on the clinical importance of "nontoxic" strains.     

Suppression of allergic immune responses to house dust mite (HDM) in rats exposed to 2,3,7,8-TCDD.

Exposure to various xenobiotics, including oxidant gases, diesel exhaust, and certain pesticides, has been reported to exacerbate pulmonary allergic hypersensitivity responses. Increased lymphocyte proliferative responses to parasite antigens or increased antibody responses to sheep erythrocyte have also been reported in rats exposed to TCDD before infection or immunization. As a result, these studies were conducted to test the hypothesis that TCDD exposure exacerbates the allergic response to house dust mite antigen. Brown Norway rats were injected, ip, with 0, 1, 10, or 30 microg TCDD/kg 7 days before intratracheal (it) sensitization to semipurified house dust mite allergen (HDM). Fourteen days later, rats were challenged with HDM and immediate bronchospasm was measured. At this time point, plus 2 and 7 days later, inflammatory cells in bronchoalveolar lavage fluid (BALF), HDM-specific IgE levels in serum, and HDM-driven cell proliferation in bronchial lymph nodes and spleen were evaluated. TCDD exposure decreased both immediate bronchoconstriction and specific IgE synthesis after the HDM challenge; 7 days later, HDM-specific IgE responses remained suppressed. Total serum IgE levels were similar in all groups. HDM challenge alone significantly increased cellular and biochemical indicators of lung injury, both of which were suppressed by TCDD exposure. The proliferative response of lymph node cells, but not of spleen cells, to HDM was also suppressed at the highest TCDD dose, although the splenic response to Concanavalin A was elevated. It appears that early events in the response to HDM are affected by TCDD exposure, since message for IL5 was dramatically reduced 2 days after sensitization, but not after challenge. We therefore conclude that TCDD exposure suppressed, rather than enhanced the development of allergic immune responses and the expression of immune-mediated lung disease.     

Molecular dosimetry of N-7 guanine adduct formation in mice and rats exposed to 1,3-butadiene.

1,3-Butadiene (BD) is a high-volume chemical used in the production of rubber and plastic. BD is a potent carcinogen in mice and a much weaker carcinogen in rats, and has been classified as a probable human carcinogen. Upon metabolic activation in vivo, it forms DNA-reactive metabolites, 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The molecular dosimetry of N-7 guanine adduct formation by these metabolites of BD in liver, lung, and kidney of B6C3F1 mice and F344 rats exposed to 0, 20, 62.5, or 625 ppm BD was studied. The adducts, racemic and meso forms of N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua), N-7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I), and N-7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II), were isolated from DNA by neutral thermal hydrolysis, desalted on solid-phase extraction cartridges, and quantitated by LC/ESI(+)/MS/MS. The number of adducts per 10(6) normal guanine bases for a given adduct was higher in mice than rats exposed to 625 ppm BD, but generally similar at lower levels of exposure. The THB-Gua adducts were the most abundant (6-27 times higher than EB-Gua) and exhibited a nonlinear exposure-response relationship. In rats, the exposure-response curves for the formation of THB-Gua adducts reached a plateau after 62.5 ppm, suggesting saturation of metabolic activation. The number of THB-Gua adducts continued to increase in mice between 62.5 and 625 ppm BD. In contrast, the less common EB-Gua adducts had a linear exposure-response relationship in both species. Combining the information from this study with previous data on BD metabolism, we were able to estimate the number of THB-Gua that resulted from DEB and EBD, and conclude that most of the THB-Gua is formed from EBD. We hypothesize that most of the EBD arises from the immediate conversion of DEB to EBD within the endoplasmic reticulum. This study highlights the need for measurements of the levels of EBD in tissues of rats and mice and for the development of a unique biomarker for DEB that is available for binding to DNA.     

Airway responses in Brown Norway rats following inhalation sensitization and challenge with trimellitic anhydride.

Trimellitic anhydride (TMA) is a cause of asthma in man. Dose-dependent TMA-specific IgE, histopathology, and airway responses after sensitization by inhalation were examined in the Brown Norway rat. Rats were exposed to 0.04, 0.4, 4, or 40 mg/m3 TMA aerosol for 10 min, once a week, over 10 weeks. All lower exposures were, subsequently, rechallenged to 40 mg/m3 TMA aerosol. All rats received a sham exposure 1 week prior to the first TMA exposure. Following the sham exposure and weekly after each TMA exposure, TMA-specific IgE and both early-phase airway response (EAR) and late-phase airway response (LAR) were measured using enhanced pause (Penh). All rats sensitized by 40 mg/m3 TMA developed specific IgE, EAR, and LAR to one or more of the challenges to 40 mg/m3 TMA. TMA of 4 mg/m3 induced a much lower, but stable, specific IgE response. EAR and LAR were observed only after a 40 mg/m3 TMA rechallenge in this group, but it was much larger than that observed in the 40 mg/m3 TMA-sensitized and challenged group. Exposure-dependent histopathological changes noted included eosinophilic granulomatous interstitial pneumonia, perivascular eosinophil infiltrates, bronchial-associated lymphoid tissue hyperplasia, and peribronchiolar plasma cell infiltrates.     

Benzo(a)pyrenediolepoxide-hemoglobin adducts and 3-hydroxy-benzo(a)pyrene urinary excretion profiles in rats subchronically exposed to benzo(a)pyrene

 The time profiles of benzo(a)pyrenediolepoxide (BaPDE)-hemoglobin (Hb) adduct formation and 3-hydroxybenzo(a)pyrene (3-OHBaP) urinary excretion were studied in male Sprague-Dawley rats exposed to daily benzo(a)pyrene (BaP) intraperitoneal doses of 1.25, 6.25, and 31.25 μmol/kg administered Tuesday to Friday for 4 consecutive weeks. Blood was withdrawn weekly, on Tuesdays, prior to dosing. Twenty four hour urine samples were collected on Mondays (following 72 h without treatment) and Thursdays. Analytes were quantified by high performance liquid chromatography (HPLC)/fluorescence. Exposure to BaP resulted in the accumulation of BaPDE-Hb adducts, reaching an average of 1.2±0.3, 8.3±1.9, and 38.2±6.1 pmol/g Hb for the 1.25, 6.25, and 31.25 μmol/kg per day doses after 4 weeks of treatment. The expected saw tooth excretion profile of 3-OHBaP was observed, with peaks on Thursdays and troughs on Mondays, and showed a progressive rise on both Mondays and Thursdays. Increase in Monday values with time suggested a possible increase in BaP body burden during exposure. To verify this aspect further, the urinary excretion kinetic of 3-OHBaP following acute intraperitoneal dosing (31.25 μmol/kg) was determined. Urine samples were collected at frequent timed intervals for up to 164 h post-dosing. Two-step elimination was observed, the second step having a half-life of 25 h, presumably linked to the slow release of BaP accumulated in fatty tissues upon repeated treatment. Therefore, it seems that 1) BaPDE-Hb adducts are good indicators of repeated exposure, 2) the difference between pre- and post-exposure 3-OHBaP urinary excretion gives a measure of recent exposure, and 3) excretion levels of this latter metabolite after a non-exposure period of sufficient duration, to allow elimination of the bulk BaP from the last dose, could reflect BaP body burden. Received: 3 November 1994/Accepted: 11 January 1995     

Changes in the contractile responses to carbachol and in the inhibitory effects of verapamil and nitrendipine on isolated smooth muscle preparations from rats subchronically exposed to Co2+ and Ni2+

Male Wistar rats were exposed to subtoxic doses of Co²⁺ or Ni²⁺, receiving Co(NO₃)₂ or NiSO₄ with drinking water for 30 days. No significant differences in the body weight and no visible changes in the behaviour of the controls and experimental animals were established. Cumulative concentration-effect curves for carbachol were obtained in ileum and trachea isolated from control and Co²⁺- or Ni²⁺-treated rats. The effect of the Ca²⁺ antagonists on the carbachol-induced contractions was studied by adding increasing concentrations of verapamil or nitrendipine to the bath solution 20 min prior to carbachol. The results showed that exposure of rats to subtoxic doses of Co(NO₃)₂ or NiSO₄ altered the contractile responses to carbachol. The changes in the pD₂ values and the shift to the left of the concentration-effect curves suggest a higher sensitivity to carbachol in preparations from the ileum of Co²⁺- or Ni²⁺-exposed rats. The tracheal strips isolated from control and heavy metal-treated rats showed a less potent sensitiveness to carbachol as compared to the ileal segments. An opposite tendency for decreased cholinergic reactivity was observed in tracheal strips from Co²⁺- and Ni²⁺-treated animals. The inhibitory effect of the Ca²⁺-antagonists on the contractility of ileal preparations from Co²⁺-treated rats increased at all concentrations of verapamil and at the highest concentration of nitrendipine, but decreased at lower concentrations of nitrendipine. The effect of verapamil on the preparations from Ni²⁺-exposed rats was unchanged or even decreased at higher verapamil concentrations. The inhibitory effect of nitrendipine on preparations from Ni²⁺-exposed rats decreased at the lowest concentration but increased at the highest concentration of the blocker. In Co²⁺- or Ni²⁺-treated tracheal preparations verapamil inhibited the contractions induced by low and medium concentrations of carbachol but increased the maximal contractile responses to high concentrations of carbachol.     

Styrene-7,8-oxide burden in ventilated, perfused lungs of mice and rats exposed to vaporous styrene.

Styrene (ST) is an important industrial chemical. In long-term inhalation studies, ST-induced lung tumors in mice but not in rats. To test the hypothesis that the lung burden by the reactive metabolite styrene-7,8-oxide (SO) would be most relevant for the species-specific tumorigenicity, we investigated the SO burden in isolated lungs of male Sprague-Dawley rats and in-situ prepared lungs of male B6C3F1 mice ventilated with air containing vaporous ST and perfused with a modified Krebs-Henseleit buffer (37 degrees C). Styrene vapor concentrations were determined in air samples collected in the immediate vicinity of the trachea. They were almost constant during each experiment. Styrene exposures ranged from 50 to 980 ppm (rats) and from 40 to 410 ppm (mice). SO was quantified from the effluent perfusate. Lungs of both species metabolized ST to SO. After a mathematical translation of the ex-vivo data to ventilation and perfusion conditions as they are occurring in vivo, a species comparison was carried out. At ST concentrations of up to 410 ppm, mean SO levels in mouse lungs ranged up to 0.45 nmol/g lung, about 2 times higher than in rat lungs at equal conditions of ST exposure. We conclude that the species difference in the SO lung burden is too small to consider the genotoxicity of SO as sufficient for explaining the fact that only mice developed lung tumors when exposed to ST. Another cause is considered as driving force for lung tumor development in the mouse.     

Immunotoxicity of aflatoxin B1 in rats: effects on lymphocytes and the inflammatory response in a chronic intermittent dosing study.

We investigated the effects of aflatoxin B1 (AFB1) on isolated splenic lymphocytes and the histo-morphologic changes in the spleens and liver of Fisher-344 male rats. Weaned animals were fed chow diets that contained 0, 0.01, 0.04, 0.4, or 1.6 ppm AFB1, using an intermittent dosing regimen (4 weeks on and 4 weeks off AFB1), for 40 weeks. An additional group of animals was fed the 1.6 ppm AFB1 diet continuously. The intermittent dosing regimen was designed to evaluate effects of cumulative dose and exposure for risk assessment comparisons. The percentages of T and B cells were affected as shown by flow cytometric analysis after the dosing cycles. The observed changes appeared to reverse or compensate to some extent after the off cycles. Lymphocytes were stimulated in culture for analysis of the production of IL-2, IL-1, and IL-6. Significantly increased production of IL-1 and IL-6 was seen in the second dosing cycle (12 weeks) and the second "off" cycle (16 weeks) at the higher doses. Inflammatory infiltrates were seen in the liver after eight weeks of continuous and intermittent dosing and were increased in size and number at 12 weeks in both 1.6 ppm dose groups correlating with the peak production of Il-1 and IL-6. We concluded that AFB1 effects on the immune system can be either stimulatory or suppressive dependent on a critical exposure window of dose and time. Immune cells in spleen such as T-lymphocytes and macrophages, both important mediators of inflammatory responses to tissue damage, were affected differently in the continuous and intermittent exposures to AFB1.     

Statistical analysis of nonmonotonic dose-response relationships: research design and analysis of nasal cell proliferation in rats exposed to formaldehyde.

Statistical analyses of nonmonotonic dose-response curves are proposed, experimental designs to detect low-dose effects of J-shaped curves are suggested, and sample sizes are provided. For quantal data such as cancer incidence rates, much larger numbers of animals are required than for continuous data such as biomarker measurements. For example, 155 animals per dose group are required to have at least an 80% chance of detecting a decrease from a 20% incidence in controls to an incidence of 10% at a low dose. For a continuous measurement, only 14 animals per group are required to have at least an 80% chance of detecting a change of the mean by one standard deviation of the control group. Experimental designs based on three dose groups plus controls are discussed to detect nonmonotonicity or to estimate the zero equivalent dose (ZED), i.e., the dose that produces a response equal to the average response in the controls. Cell proliferation data in the nasal respiratory epithelium of rats exposed to formaldehyde by inhalation are used to illustrate the statistical procedures. Statistically significant departures from a monotonic dose response were obtained for time-weighted average labeling indices with an estimated ZED at a formaldehyde dose of 5.4 ppm, with a lower 95% confidence limit of 2.7 ppm. It is concluded that demonstration of a statistically significant bi-phasic dose-response curve, together with estimation of the resulting ZED, could serve as a point-of departure in establishing a reference dose for low-dose risk assessment.     

Motor impairment in rats exposed to PCBs and methylmercury during early development.

Epidemiological and laboratory studies indicate that polychlorinated biphenyls (PCBs) and methyl mercury (MeHg) may have additive or interactive adverse effects on nervous system function. Prior studies have shown that high doses of MeHg target the cerebellum and impair balance and coordination, but the effects of PCBs on cerebellar function were unknown. In addition, the combined effects of PCBs and MeHg on cerebellar function have not been studied previously. Therefore, we investigated the effects of developmental exposure to PCBs, MeHg, or PCBs + MeHg on three motor tasks that involve cerebellar functions. Female Long-Evans rats were exposed to MeHg (0.5 ppm in drinking water), PCBs (6-mg/kg/d Aroclor 1254), PCBs + MeHg, or vehicle only beginning 4 weeks prior to breeding, through pregnancy, and continuing through postnatal day (PND) 16. Starting at approximately PND 60, one male and one female from each litter were tested on three motor tasks that involve cerebellar function. PCB + MeHg-exposed rats were impaired relative to the controls on a task requiring them to traverse a rotating rod. Rats exposed to PCBs alone were also somewhat impaired relative to the controls, whereas MeHg-exposed rats were not significantly different from the controls. There were no statistically significant deficits related to PCB or MeHg exposure on a vertical rope-climbing test or a parallel bar test. Our results demonstrate that the possibility of additive neurotoxic effects of PCBs and MeHg needs to be seriously considered.     

Altered mammary gland development in male rats exposed to genistein and methoxychlor.

Genistein (GE) is a prevalent phytoestrogen whose presence in human and animal foods may affect biological actions of synthetic endocrine active compounds. We have previously reported that in utero and lactational exposure to high doses of GE or the endocrine active pesticide methoxychlor (MXC) caused mammary epithelial proliferation in 21-day-old male rats. Combined exposure to GE and MXC resulted in significant feminization of the male mammary glands. The goals of the current study were to evaluate mammary responses to GE and MXC at the adult stage and investigate relevant mechanisms. Following in utero, lactational exposure (through maternal diet), and direct dietary exposure, the inguinal mammary gland of male rats (90 days of age) was found to exhibit significant morphological alterations in the groups treated with GE and/or MXC compared to the control. GE exposure (at 300 and 800 ppm concentrations) caused lobular enlargement and epithelial proliferation, whereas MXC exposure (800 ppm) led to ductal elongation and lobular enlargement. Combining the two treatments caused prominent proliferation of both ducts and alveoli; secretory material was seen in readily recognizable alveolar lumens, which are absent in untreated male mammary. We also surveyed gene expression in the mammary tissue using a cDNA microarray and evaluated relevant protein factors. The results indicated that the treatment effects are likely due to interactions between steroid hormone receptor-mediated signals and growth factor-driven cellular pathways. The distinctive responses associated with the GE+MXC combination were likely linked to enhanced actions of insulin-like growth factor 1 and related downstream pathways.     

Lung tumor development in mice exposed to tobacco smoke and fed beta-carotene diets.

In human clinical trials it was found that the putative chemopreventive agent beta-carotene not only failed to protect active smokers against the carcinogenic action of tobacco smoke, but actually increased their risk of developing lung cancer. In preclinical animal studies, beta-carotene had been effective against some chemically induced cancers, but not against tumors in the respiratory tract. We exposed male strain A/J mice to tobacco smoke at a concentration of 140 mg/m(3) of total suspended particulate matter, 6 h a day, 5 days a week, for either 4 or 5 months, followed by a recovery period in air for 4 or 5 months, or for 9 months without recovery period. beta-carotene was added in the form of gelatin beadlets to the AIN-93G diet either during or following tobacco smoke exposure at concentrations of 0.005, 0.05 and 0.5%. In the supplement-fed animals, plasma and lung levels of beta-carotene were higher than they were in animals fed control diets. Exposure to tobacco smoke increased rather than decreased plasma beta-carotene levels, but had no significant effect on lung levels. After 9 months, lung tumor multiplicities and incidence were determined. Tobacco smoke increased both lung tumor multiplicities and incidences, but beta-carotene failed to modulate tumor development under all exposure conditions. Animal studies in a model of tobacco smoke carcinogenesis would thus have predicted the absence of any beneficial effects of beta-carotene supplementation in current or former smokers, but would have failed to anticipate the increase in lung cancer risk.     

Acute hemorrhagic myocardial necrosis and sudden death of rats exposed to a combination of ephedrine and caffeine.

Because of possible side effects of herbal medicines containing ephedrine and guarana-derived caffeine, including increased risk of stroke, myocardial infarction, and sudden death, the Food and Drug Administration recently banned the sale of ephedra-containing products, specifically over-the-counter dietary supplements. We report cardiac in 7- and 14-week-old male F344 rats exposed by gavage to ephedrine(25 mg/kg) and caffeine (30 mg/kg) administered in combination for one or two days. The ephedrine-caffeine dosage was approximately 12- and 1.4-fold, respectively, above average human exposure, based on a mg/m2 body surface-area comparison. Several (5/7) of the exposed 14-week-old rats died or were sacrificed in extremis 4-5 h after the first dosing. In these hearts, changes were observed chiefly in the interventricular septum but also left and right ventricular walls. Massive interstitial hemorrhage, with degeneration of myofibers, occurred at the subendocardial myocardium of the left ventricle and interventricular septum. Immunostaining for cleaved caspase-3 and hyperphosphorylated H2A.X, a histone variant that becomes hyperphosphorylated during apoptosis, indicated multifocal generalized positive staining of degenerating myofibers and fragmenting nuclei, respectively. The Barbeito-Lopez trichrome stain revealed generalized patchy yellow myofibers consistent with degeneration and/or coagulative necrosis. In ephedrine-caffeine-treated animals terminated after the second dosing, foci of myocardial degeneration and necrosis were already infiltrated by mixed inflammatory cells. The myocardial necrosis may occur secondarily to intense diffuse vasoconstriction of the coronary arterial system with decreased myocardial perfusion. Our work shows the direct relationship between combined ephedrine and caffeine exposure and cardiac pathology.     

氰戊菊酯染毒大鼠肺灌洗液中细胞与生化效应

给大鼠一次气管注入氰戊菊酯悬液(FS)以及中毒室内吸入氰戊菊酯(FA),通过分析BALF中细胞与生化组分评价肺脏的毒性反应。结果表明PAM_s数下降,PMN数、Alb和NANA含量增加,LDH、ALP和ACP活性升高,提示氰戊菊酯引起肺细胞生物膜损伤、弥漫性肺泡炎和肺水肿。氰戊菊酯的阈剂量和阈浓度分别为0.93 mg/kg和200 mg/m~3,阈下剂量和阈下浓度为0.19 mg/kg和40 mg/m~2。研究结果为探讨氰戊菊酯的肺脏毒性及其机理,为制订其卫生标准提供依据。     

Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.     

Kinetic modeling of beta-chloroprene metabolism: I. In vitro rates in liver and lung tissue fractions from mice, rats, hamsters, and humans.

Beta-chloroprene (2-chloro-1,3-butadiene, CD) is carcinogenic by inhalation exposure to B6C3F1 mice and Fischer F344 rats but not to Wistar rats or Syrian hamsters. The initial step in metabolism is oxidation, forming a stable epoxide (1-chloroethenyl)oxirane (1-CEO), a genotoxicant that might be involved in rodent tumorigenicity. This study investigated the species-dependent in vitro kinetics of CD oxidation and subsequent 1-CEO metabolism by microsomal epoxide hydrolase and cytosolic glutathione S-transferases in liver and lung, tissues that are prone to tumor induction. Estimates for Vmax and Km for cytochrome P450-dependent oxidation of CD in liver microsomes ranged from 0.068 to 0.29 micromol/h/mg protein and 0.53 to 1.33 microM, respectively. Oxidation (Vmax/Km) of CD in liver was slightly faster in the mouse and hamster than in rats or humans. In lung microsomes, Vmax/Km was much greater for mice compared with the other species. The Vmax and Km estimates for microsomal epoxide hydrolase activity toward 1-CEO ranged from 0.11 to 3.66 micromol/h/mg protein and 20.9 to 187.6 microM, respectively, across tissues and species. Hydrolysis (Vmax/Km) of 1-CEO in liver and lung microsomes was faster for the human and hamster than for rat or mouse. The Vmax/Km in liver was 3 to 11 times greater than in lung. 1-CEO formation from CD was measured in liver microsomes and was estimated to be 2-5% of the total CD oxidation. Glutathione S-transferase-mediated metabolism of 1-CEO in cytosolic tissue fractions was described as a pseudo-second order reaction; rates were 0.0016-0.0068/h/mg cytosolic protein in liver and 0.00056-0.0022 h/mg in lung. The observed differences in metabolism are relevant to understanding species differences in sensitivity to CD-induced liver and lung tumorigenicity.     

Alterations in gene expression and testosterone synthesis in the testes of male rats exposed to perfluorododecanoic acid.

Perfluorododecanoic acid (PFDoA, C12), a synthetic perfluorinated chemical containing 12 carbons, has broad industrial applications and has been detected in sera from humans and other animals; however, few reports have addressed the effects of PFDoA exposure on male reproduction. In the present study, the effects of PFDoA exposure on testes ultrastructure, testosterone levels, and steroidogenic gene expression were investigated. Male rats were orally dosed for 14 days with 1, 5, or 10 mg PFDoA/kg/day or with vehicle. Absolute testis weight was diminished at the highest dose while relative testes weight was markedly increased at doses of 5 and 10 mg/kg/day. Total serum cholesterol levels were significantly increased at the highest dose. While luteinizing hormone was significantly decreased at the highest dose, testosterone was markedly decreased at doses of 5 and 10 mg PFDoA/kg/day. Serum levels of follicle-stimulating hormone were not significantly affected by PFDoA, and estradiol levels were markedly decreased only at 5 mg/kg/day. Leydig cells, Sertoli cells, and spermatogenic cells from rats that received 5 or 10 mg PFDoA/kg/day, exhibited apoptotic features including dense irregular nuclei, condensed chromatin, ill-defined nuclear membranes, and abnormal mitochondria. PFDoA exposure resulted in significant declines in mRNA expression of several genes involved in cholesterol transport and steroid biosynthesis at doses of 5 and 10 mg PFDoA/kg/day, while the gene expression of luteinizing hormone receptor and aromatase was not significantly changed. Our results demonstrate that PFDoA affects the reproduction function of male rats via alterations in steroidogenesis genes, testosterone levels, and testes ultrastructure.     

Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: effects of dose, route, and diet.

The azoxymethane (AOM) model has been widely used to investigate the pathology and genetics of colorectal cancer in rodents. However, there has been wide variation in treatment regimes, making it difficult to compare across studies. Consequently, standardizing AOM treatment and identifying sources of experimental variation would allow better comparisons across studies. In order to establish an optimal dosing regime for detecting experiment-dependent differences in tumorigenesis, we performed a dose curve analysis using AKR/J, SWR/J, and A/J mouse strains previously reported to vary widely in susceptibility to AOM. Although intraperitoneal or subcutaneous administration, but not in utero exposure, resulted in similar levels of tumor induction, significant dose- and strain-dependent effects of AOM were observed. No sex-dependent differences were observed. Increasing the number of treatments uncovered a significant strain-dependent effect on tumor promotion, independent of susceptibility to tumor initiation. Similarly, we used C57BL/6J and DBA/2J intercrosses to demonstrate that small diet modifications can significantly alter AOM-induced tumorigenesis in a background-dependent manner. These results provide experimental support for a standardized AOM treatment and for the importance of controlling both genetic and non-genetic factors when using this model.