Effects of acrylamide exposure on serum hormones, gene expression, cell proliferation, and histopathology in male reproductive tissues of Fischer 344 rats.

Acrylamide (AA) is a reactive monomer used in many technological applications, but it is the incidental formation during cooking of common starchy foods that leads to pervasive human exposure, typically in the range of 1 µg/kg body weight (bw)/day (d). AA is carcinogenic in multiple organs from both sexes of several rodent models and a consistent body of evidence points to a genotoxic mechanism based on metabolism to a DNA-reactive epoxide, glycidamide (GA). In F344 rats, tumorigenesis occurs in several hormonally regulated tissues (thyroid, mammary gland, and peri-testicular mesothelium), which has prompted speculation about endocrine dysregulation as a possible mechanism. The present study evaluated the effects of a 14 d exposure to AA administered through the drinking water on reproductive tissues and the hypothalamic-pituitary-testes (HPG) axis in male F344 rats. The doses selected encompass a range from approximately 2.5 mg/kg bw/d, which is carcinogenic after lifetime exposure, to 50 mg/kg bw/d, a maximally tolerable dose that causes hind limb paralysis. AA caused significant changes in serum hormones, histopathology, testicular gene expression, and cell proliferation, especially at the highest dose. Despite strong evidence for activation of the HPG axis subsequent to decreases in testosterone levels, and histopathological changes associated with significant effects on Leydig and germ cells, with concomitant mRNA expression changes, the precise mechanism(s) for AA-induced testicular toxicity remains unclear; however, the absence of evidence for increased proliferation of the peri-testicular mesothelium (Ki-67 immunoreactivity) does not support hormonal dysregulation as a contributing factor to the predisposition of this tissue to the carcinogenic effects of AA.

The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male Fischer 344 rats.

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Trichloroethylene, trichloroacetic acid, and dichloroacetic acid: do they affect eye development in the Sprague-Dawley rat?

Maternal exposure to high doses of trichloroethylene (TCE) and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), has been implicated in eye malformations in fetal rats, primarily micro-/anophthalmia. Subsequent to a cardiac teratology study of these compounds (Fisher et al. 2001, Int. J. Toxicol. 20:257-267), their potential to induce ocular malformations was examined in a subset of the same experimental animals. Pregnant, Sprague-Dawley Crl:CDR BR rats were orally treated on gestation days (GDs) 6 to 15 with bolus doses of either TCE (500 mg/kg/day), TCA (300 mg/kg/day), DCA (300 mg/kg/day), or all-trans retinoic acid (RA; 15 mg/kg/day). The heads of GD 21 fetuses were not only examined grossly for external malformations, but were sectioned using a modified Wilson's technique and subjected to computerized morphometry that allowed for the quantification of lens area, globe area, medial canthus distance, and interocular distance. Gross ocular malformations were essentially absent in all treatment groups except for the RA group in which 26% of fetuses exhibited micro-/anophthalmia. Using the litter as the experimental unit of analysis, lens area, globe area, and interocular distance were statistically significantly reduced in the DCA treatment group. Statistically significant reductions in lens and globe areas also occurred in the RA treatment group, all four ocular measures were reduced in the TCA treatment group but none significantly so, and TCE was without effect. Because DCA, TCA, and RA treatments were associated with significant reductions in fetal body weight (bw), data were also statistically analyzed after bw adjustment. Doing so dramatically altered the results of treatment group comparisons, but the severity of bw reduction and the degree of change in ocular measures did not always correlate. This suggests that bw reduction may not be an adequate explanation for all the changes observed in ocular measures. Thus, it is unclear whether DCA specifically disrupted ocular development even under these provocative exposure conditions. Clearly, however, if TCE is capable of disrupting ocular development in the Sprague-Dawley rat, a higher dose than that employed in the present study is required.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Polycystic kidney disease induced in F(1) Sprague-Dawley rats fed para-nonylphenol in a soy-free, casein-containing diet.

para-Nonylphenol (NP; CAS #84852-15-3), an alkylphenol with a 9-carbon olefin side chain, is widely used in the manufacture of nonionic surfactants, lubricant additives, polymer stabilizers, and antioxidants. Due to its wide commercial use and putative endocrine activity in humans and wildlife, the NTP elected to assess its effects on reproduction in multigenerational studies. To avoid known estrogenic activity of phytoestrogens in soy and alfalfa, a soy- and alfalfa-free, casein-containing diet was used in a range-finding study to determine the doses of NP to be tested further. NP was administered to Sprague-Dawley rats in the diet at 0, 5, 25, 200, 500, 1000, or 2000 ppm to F(0) dams beginning on gestation-day 7. The F(1) pups were weaned at postnatal day (PND) 21, and their exposure via diet was continued at the same dose level as their respective dams. Pup weights from birth through weaning were not significantly different from controls in any dose group, but the average weight of both sexes was significantly less compared to controls, beginning with the PND 28 weighing. The F(1) rats were sacrificed on PND 50 (n = 15, 3 pups of each sex from 5 litters for all dose groups). Terminal body weights of males and females in the 2000-ppm dose group were 74% and 85% of controls, respectively. Severe polycystic kidney disease (PKD) was present in 100% of the 2000 ppm-exposed male and female rats. At 1000 ppm, 67% of males and 53% of females had mild to moderate PKD versus none of either sex in the control and lower-dose groups. The no-adverse-effect level (NOAEL) for PKD was determined to be 500 ppm. Previous studies with comparable duration and route of exposure, but using soy-containing diets, reported either no or only mild PKD at 2000 ppm NP. We conclude that the renal toxicity of NP is highly dependent on the diet on which the animals are maintained. The potential interaction of diet and test compounds on nonreproductive as well as reproductive endpoints should be considered when contemplating the use of special diets formulated to minimize exogenous "hormone" content for the study of the effects of putative endocrine disruptive chemicals.

Trichloroethylene, trichloroacetic acid, and dichloroacetic acid: do they affect fetal rat heart development?

Trichloroethylene (TCE), trichloroacetic acid (TCA), and dichloroacetic acid (DCA) are commonly found as groundwater contaminants in many regions of the United States. Cardiac birth defects in children have been associated with TCE, and laboratory studies with rodents report an increased incidence of fetal cardiac malformations resulting from maternal exposures to TCE, TCA, and DCA. The objective of this study was to orally treat pregnant CDR(CD) Sprague-Dawley rats with large bolus doses of either TCE (500 mg/kg), TCA (300 mg/kg), or DCA (300 mg/kg) once per day on days 6 through 15 of gestation to determine the effectiveness of these materials to induce cardiac defects in the fetus. All-trans retinoic acid (RA) dissolved in soybean oil was used as a positive control. Soybean oil is commonly used as a dosing vehicle for RA teratology studies and was also used in this study as a dosing vehicle for TCE. Water was used as the dosing vehicle for TCA and DCA. Fetal hearts were examined on gestation day (GD) 21 by an initial in situ, cardiovascular stereomicroscope examination, and then followed by a microscopic dissection and examination of the formalin-fixed heart. The doses selected for TCA and DCA resulted in a modest decrease in maternal weight gain during gestation (3% to 8%). The fetal weights on GD 21 in the TCA and DCA treatment groups were decreased 8% and 9%, respectively, compared to the water control group and 21% in the RA treatment group compared to soybean oil control group. The heart malformation incidence for fetuses from the TCE-, TCA-, and DCA-treated dams did not differ from control values on a per fetus or per litter basis. The rate of heart malformations, on a per fetus basis, ranged from 3% to 5% for TCE, TCA, and DCA treatment groups compared to 6.5% and 2.9% for soybean oil and water control groups. The RA treatment group was significantly higher with 33% of the fetuses displaying heart defects. For TCE, TCA, and DCA treatment groups 42% to 60% of the litters contained at least one fetus with a heart malformation, compared to 52% and 37% of the litters in the soybean oil and water control groups. For the RA treatment group, 11 of 12 litters contained at least one fetus with a heart malformation. Further research is needed to quantify the spontaneous rates of heart defects for vehicle control rats and to explain the disparity between findings in the present study and other reported findings on the fetal cardiac teratogenicity of TCE, TCA, and DCA.

A subchronic exposure to trichloroethylene causes lipid peroxidation and hepatocellular proliferation in male B6C3F1 mouse liver.

The common groundwater contaminant trichloroethylene (TCE), when given by oral gavage, can produce free radical species during metabolism. Furthermore, TCE end-stage metabolites, trichloroacetic acid and dichloroacetic acid, cause lipid peroxidation in mouse liver. The time courses of lipid peroxidation, free radical generation, and 8-hydroxydeoxyguanosine (8OHdG) formation were used to assess the level of oxidative stress in the liver of B6C3F1 mice dosed orally once daily, 5 days a week for 8 weeks at 0, 400, 800, and 1200 mg/kg TCE in corn oil. Peroxisomal proliferation, cell proliferation, and apoptosis were evaluated at selected times during the study. Lipid peroxidation, as measured by thiobarbituric acid-reactive substances (TBARS), was significantly elevated at the two highest dose levels of TCE on days 6 through 14 of the study. 8OHdG levels were statistically significant in the 1200 mg/kg/day group on days 2, 3, 10, 28, 49, and 56 only. The highest measured free radical load, 307% of oil control, occurred at day 6. A significant increase in cell and peroxisomal proliferation was observed during the same time period in the 1200 mg/kg/day group. Necrosis or an increase in apoptosis was not observed at any dose. The temporal relationship between oxidative stress and cellular response of proliferation, both of which occur and resolve within the same relative time period, suggests that TCE-induced mitogenesis may result from alteration in the liver microenvironment which offers a selective advantage for certain hepatocyte subpopulations.

Dissimilar characteristics of N-methyl-N-nitrosourea-initiated foci and tumors promoted by dichloroacetic acid or trichloroacetic acid in the liver of female B6C3F1 mice.

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are metabolites of the industrial solvent and environmental contaminant trichloroethylene (TCE), as well as contaminants of chlorinated drinking water. Human exposure to these chemicals is of concern as all three have been shown to increase liver tumor incidence in mice. Differences in dose-response curves, progression to cancer, and postexposure regression of lesions suggest that TCA and DCA work through different mechanisms. The purpose of this study was to further characterize the proliferative hepatocellular lesions promoted by TCA and DCA using biomarkers of cell growth, differentiation, and metabolism in liver sections to better delineate the distinctions in the mechanism of the two chloroacetates. Fifteen-day-old female mice were initiated with 25 mg/kg N-methyl-N-nitrosourea. The initiated mice were administered DCA or TCA (20.0 mmol/L) in drinking water from age 49 days until euthanasia at age 413 days. The pathologic assessment showed that the foci of altered hepatocytes and tumors occurring in the animals promoted with DCA were eosinophilic and positive immunohistochemically for TGF-alpha, c-jun, c-myc, CYP 2E1, CYP 4A1, and glutathione S-transferase-pi (GST-pi). The DCA lesions also were essentially negative for c-fos and TGF-beta, but nontumor hepatocytes were consistently TGF-beta-positive. In contrast, tumors promoted by TCA were predominantly basophilic, lacked GST-pi, and stained variably; usually, more than 50% of the tumor hepatocytes were essentially negative for the other biomarkers. This study demonstrates some striking differences in certain molecular biomarkers of cell growth, differentiation, and metabolism between DCA and TCA. The results also suggest some potential growth signal transduction pathways that may contribute to the DCA promotion of tumors, further support the premise that these two chloroacetates promote hepatocarcinogenesis in different ways, and provide a rational basis for a similar comparison with TCE. Such a comparison should give some insight as to whether DCA, TCA, or both are playing a significant role in the murine liver carcinogenesis of the parent compound, TCE.

Oncogenic potential of inhaled hydrazine in the nose of rats and hamsters after 1 or 10 1-hr exposures.

Hydrazine (N2H4) is used as a fuel for missiles and standby power systems of operational military aircraft. Maintenance of missiles and aircraft may result in accidental human exposure to high concentrations for brief periods of time. The purposes of this study were to assess the oncogenic potential of N2H4 in rats and male hamsters exposed to a high concentration of N2H4 for repeated short exposures and to investigate the relationships of acute and subchronic effects of N2H4 to nasal tumorigenesis. In phase 1 (acute and subchronic) and Phase 2 (lifetime experiments, groups of male and female Fischer 344 rats and male Syrian golden hamsters were exposed by inhalation to 0, 75 (Phase 2 only), or 750 ppm N2H4 for 1 (acute) or 10 (subchronic) 1-hr weekly exposures. Rodents were euthanized 24 hr after exposures 1 and 10 and 24 to 30 months poststudy initiation. Significant reductions in body weight were observed in N2H4-treated rodents compared to controls during the exposure interval. No hydrazine-induced mortality was detected. Histopathologic examination after the acute and subchronic exposures revealed degeneration and necrosis of transitional, respiratory, and olfactory epithelia in the anterior nose and, in rats exposed subchronically, squamous metaplasia of the transitional epithelium. Minimal to mild rhinitis resulted from N2H4 exposures. Apoptosis was observed in olfactory and squamous metaplastic transitional epithelium. Lesions occurred at sites reportedly having high air-flow and generally appeared to be more severe in the anterior portion of the nose. By 24 months, the squamous metaplastic transitional epithelium reverted back to normal-appearing transitional epithelium. By 24+ months, low incidences (sexes combined) of hyperplasia (5/194, 2.6%) and neoplasia (11/194, 5.7%) were detected, principally in the transitional epithelium of the 750 ppm N2H4-treated rats. A similar incidence of hyperplasia (2/94, 2%) and neoplasia (5/94, 5.3%) was detected in the high-exposure group of hamsters. The location and type of N2H4-induced proliferative lesions were similar to those reported in a chronic N2H4-exposure study (5.0 ppm x 6 hr/day x 5 days/week for 1 year) conducted in our laboratory, but the chronic study had much higher incidences (rats, sexes combined: hyperplasia 15.5% vs 2.6% and polypoid adenoma 44.6% vs 5.2%). The product (CD) of concentration + time was the same (750 ppm hours) for the high-dose groups for both studies, but the duration of exposure was 150 x longer and the concentration was 150 x lower in the chronic study.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxic effects of butylated triphenyl phosphate-based hydraulic fluid and tricresyl phosphate in female F344 rats.

Triaryl phosphates, including tricresyl phosphate (TCP and butylated triphenyl phosphates (BTP), are used in the commercial manufacture of plastics, lubricants, and hydraulic fluids. Recent reports implicate these compounds as endocrine and reproductive toxicants that can cause cholesteryl lipidosis in adrenocortical (AC) and ovarian interstitial (OI) cells, suggesting altered metabolism of steroid hormones or cholesterol or of both. We investigated potential mechanisms of BTP and TCP toxicity to determine if there were functional abnormalities of the adrenal cortex or ovary. Groups of intact (nine or 12) and ovariectomized (six) female F344 rats, 10-12 weeks of age, received 0, 0.4 g/kg TCP, or 1.7 g/kg BTP in sesame oil vehicle or 1.7 g/kg neat BTP for 20, 40, or 60 days. All rats administered BTP and TCP developed cholesteryl lipidosis in AC and OI cells; the TCP-treated group was most severely affected. Serum concentrations of androstenedione and corticosterone were unchanged, but estradiol levels were significantly (P < or = 0.05) elevated in BTP- and TCP-treated groups (14.5 times and 37.5 times greater than controls, respectively). Vaginal cytology revealed that BTP- but not TCP-treated females had abnormal reproductive cycles that were significantly prolonged in diestrus (3 times greater than control). There were significant elevations in serum total cholesterol (TCP-treated group was 1.3 times greater than controls), low-density lipoprotein (TCP-treated group was 1.8 times greater than controls), alanine transaminase (BTP-treated group was 2 times greater than controls), and albumin (a major serum estradiol-binding protein; BTP-treated group was 4.6 g/dl vs. 3.6 g/dl for controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Pathologic effects of butylated triphenyl phosphate-based hydraulic fluid and tricresyl phosphate on the adrenal gland, ovary, and testis in the Fischer-344 rat.

Triaryl phosphates including tricresyl phosphate (TCP) and butylated triphenyl phosphates (BTPs) are used in the commercial manufacture of plastics, lubricants, and hydraulic fluids. Recent reports implicate these compounds as endocrine and reproductive toxicants in rodents. The objectives of this study were to develop and characterize a rat model to investigate the mechanism(s) of toxicity of triaryl phosphate-based hydraulic fluids and to elucidate potential mechanistic pathways of toxicity through studies of structural/functional relationships. Groups of male and female rats received daily oral doses of either sesame oil alone or 1.7 g/kg of BTP or 0.4 g/kg TCP in sesame oil vehicle or 2.8 g/kg neat BTP for 20, 40, and 60 days. Light microscopic, morphometric, ultrastructural, and histochemical studies revealed hypertrophy and cholesteryl lipidosis of adrenocortical (both sexes) and ovarian interstitial cells that were progressive with duration of exposure. Minimal degeneration was observed in the adrenal cortex and ovary. TCP caused the most severe lesions in both the adrenal gland and ovary, but the morphologic and histochemical changes produced were similar for both compounds, suggesting a common mechanism of toxicity. Decreased testicular weight and degeneration of seminiferous tubules were detected only in TCP-treated rats. The Fischer-344 rat model using TCP and BTP administered by gavage is a valuable system to study mechanisms of endocrine and reproductive toxicity induced by triaryl phosphate-based hydraulic fluids.

Reproductive toxicity of butylated triphenyl phosphate and tricresyl phosphate fluids in F344 rats.

The effects of tricresyl phosphate (TCP) and butylated triphenyl phosphate (BTP)-based hydraulic fluid on reproduction were studied in F344 rats using a modification of the National Toxicology Program's Continuous Breeding Protocol. Groups of breeding pairs received single daily oral doses of an equal volume of either 0, 0.6, 1.0 g BTP/kg or 0.4 TCP/kg in sesame oil or 1.7 g neat BTP/kg for up to 135 days. A naive control group allowed to breed, but not dosed or handled daily, demonstrated that daily dosing and handling of the rats had no effect on reproduction. The fertility index and number of litters born were significantly decreased in rats exposed to 1.0 and 1.7 g BTP/kg and 0.4 g TCP/kg. The number of pups per litter was significantly decreased in the TCP group. A crossover mating experiment using 0.4 g TCP/kg/day and 1.0 g BTP/kg/day groups, each mated with vehicle controls, demonstrated that TCP caused 100% infertility in male rats but did not affect reproduction in females. BTP caused a significant decline in reproduction in female rats characterized by low mating and fertility indices, decreased number of litters, and abnormal estrous cycles. Fertility was decreased in the BTP-dosed male rats. Both sexes of rats in the crossover experiment with TCP and BTP had significant decreases in terminal body weights and increases in adrenal gland and liver weights. Only TCP-dosed male rats had significantly decreased testicular and epididymal weights.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenesis of cholesteryl lipidosis of adrenocortical and ovarian interstitial cells in F344 rats caused by tricresyl phosphate and butylated triphenyl phosphate.

Triaryl phosphates including tricresyl phosphate (TCP) and butylated triphenyl phosphate (BTP) are organophosphates used in the commercial manufacture of plastics, lubricants, and hydraulic fluids. Rat steroidogenic tissues such as adrenocortical (AC), ovarian interstitial (OI), and Leydig cells use an intracellular pathway to store cholesterol (substrate for biosynthesis of steroid hormones) as cholesteryl ester (CE). This pathway and the pathway for uptake of serum cholesterol are less used in Leydig cells of the adult male rat, resulting in a lower CE pool. BTP and TCP caused cholesteryl lipidosis in steroid hormone-synthesizing AC and OI, but not Leydig cells in the adult rat. The objectives of this study were to determine if the administration of triaryl phosphate fluids caused a defect in the cholesterol storage pathway of AC and OI cells and to determine the mechanism of action. Female rats received daily oral doses of 0 or 0.4 g/kg TCP in sesame oil vehicle or 1.7 g/kg neat BTP for 40 days. Adrenal glands from both treatment groups and ovaries from TCP-treated rats were heavier than controls. Microscopic and biochemical studies revealed cholesteryl lipidosis composed of CE in the adrenal glands and ovaries in BTP- and TCP-treated rats with the latter group affected most severely. The activity of neutral CE hydrolase (nCEH), an enzyme that converts CE to cholesterol in the uptake and storage pathways, also was inhibited most in the TCP-treated group (97% inhibition compared to that of control). The activity of acyl coenzyme A:cholesterol acyl transferase, an enzyme that esterifies cholesterol to make CE, was depressed 27% compared to that of control adrenal glands of the TCP group, resulting in elevated intracellular cholesterol levels in AC cells. An inhibition of nCEH in the storage and uptake pathways by triaryl phosphates most likely resulted in the striking accumulation of CE in cytoplasmic lipid droplets of AC and OI cells in F344 rats.

Japanese encephalitis virus in Bangkok: factors influencing vector infections in three suburban communities.

An unexpected outbreak of Japanese encephalitis (JE) in Bangkok in 1985 led us to investigate the vector ecology of urban JE from January 1986 to June 1987 at three suburban sites that displayed a wide range of factors imputed to influence JE transmission. Culex tritaeniorhynchus Giles and Cx. gelidus Theobald, suspected vectors, comprised 71-96% of all mosquitoes collected by CO2-baited CDC traps at the three sites. Mean of mosquito abundance per two trap-nights per month ranged from 28 to 5,728 mosquitoes at the sites of lowest and highest abundance, respectively. Cx. tritaeniorhynchus yielded more JE isolates (n = 16) than Cx. gelidus (n = 7), but the minimum infection rates of the two species (number of JE isolates per 1,000 mosquitoes tested; MIR, 0.17 and 0.47, respectively) were comparable and covaried with vector abundance. Moreover, the proportion of sentinel pigs that had JE antibodies generally increased proportionately with vector abundance at the sites. Vector abundance was high in monsoon (May-October), moderate in transition (March-April and November-December), and low in dry (January-February) seasons. Mosquitoes collected in monsoon seasons yielded 96% of the JE isolates, whereas 4 and 0% of the isolates were obtained from transition and dry season collections, respectively. More pigs seroconverted in monsoon and transition seasons than in dry seasons. Indices of JE transmission activity (vector abundance, pig seroconversions, and MIRs) increased proportionately with rainfall. Despite higher indices at the site of greatest vector abundance than elsewhere, the risk of human infection appeared greatest at the site with moderate vector abundance because of its greatest human population density.

A longitudinal study of Japanese encephalitis in suburban Bangkok, Thailand.

A one-year study of Japanese encephalitis (JE) in a small focus of transmission was conducted in suburban Bangkok in 1985. Monthly data were collected on weather, vector density, sentinel pig and chick JE antibody seroconversions, and epidemiology as related to human JE cases. The primary vector species were found to be Culex gelidus and Culex tritaeniorhynchus; from which one isolate each was obtained in March and June, respectively. Pig JE antibody seroconversion peaked in April (the hottest month), with secondary peaks following in July and December. Chick seroconversions were found only in June and July. Human cases (7) in the primary focus occurred from May-July, and started 2 months following the finding of the first JEV isolate in mosquitoes and 1 month following mass JEV seroconversion in pigs. Overall, the attack rate in the focus (0.83/10(5] was greater than 4 times that of the rest of Bangkok (0.19/10(5]. Attack rates were highest in 0-9 and 10-19 year-old groups, respectively. Indications are that JEV is transmitted to humans in Bangkok at least 10 out of 12 months per year, but that cases are concentrated in the May to July period.

The pathology of lymphoreticular and genital tissues of silvered leaf monkeys (Presbytis cristata) experimentally infected with Wuchereria bancrofti.

The discovery of the silvered leaf monkey, Presbytis cristata, as a suitable experimental host for the human filarial parasite, Wuchereria bancrofti, opened the door for major advances in our understanding of the disease caused by this parasite. To study the pathogenesis of bancroftian filariasis in this model, 15 adult P. cristata which had been experimentally infected with 250 infective third-stage larvae of the parasite were examined. After inoculation with larvae, the monkeys were maintained under study for periods of 2 to 3 years, at which time all had achieved patent infections. At necropsy, Wuchereria-induced macroscopic lesions were not detected in experimentally infected monkeys. Microscopic findings included nematodiasis, microfilariasis, lymphadenitis, lymphangiectasis, perilymphangitis, splenitis, orchitis, periorchitis, epididymitis and funiculitis. Sections of normal adult worms were most often found in lymphatic vessels near lymph nodes, or in the lymphatics of the male genital system. These worms caused microfiliariasis in some regional lymph nodes. Inflammatory responses to filariae were most prominent in proximity to degenerated and dead worms, whereas intact, normal appearing adult worms elicited only minimal cellular response.

Neurovirulence detection of dengue virus using rhesus and cynomolgus monkeys.

The results of a comparative study of neurovirulence of dengue type 1 virus in two species of Old World monkeys, viz. rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are reported. In the present study, parental dengue type 1 (16007) and its vaccine viruses were tested by intrathalamic, intramuscular and intraspinal injections in these two species of monkey. Both species of monkeys inoculated with parental dengue type 1 virus developed neurovirulence-type lesions which were graded as minimal (V-1) and occasionally mild (V-2, in cynomolgus monkeys) in severity. The antibody response to either parental or vaccine virus was slightly less in rhesus monkeys than in cynomolgus inoculated with these strains. This comparative study possibly establishes the cynomolgus monkey as a suitable test model to replace the rhesus monkey for neurovirulence testing of dengue-1 vaccine intended for use in humans.

The neurovirulence of flaviviruses in crab-eating monkeys (Macaca fascicularis).

The neurovirulent properties of attenuated dengue-2 and yellow fever (YF) vaccines, dengue-2 (DEN-2) and Japanese encephalitis (JE) viruses were studied in crab-eating monkeys (Macaca fascicularis). Number of central nervous system sites (as proportion affected) with neurovirulence (NV) lesions were compared. The results indicate that these monkeys reliably developed NV-lesion when inoculated with either JE or YF vaccine viruses (87%). NV-lesions occurred in a minority when inoculated with DEN-2 vaccine virus, were of minimal severity (9%), were probably biologically insignificant, and were of equal or less severity than lesions produced by its parental virus (10%).

Alterations in alveolar clearance after 4-ipomeanol-induced necrosis of Clara and ciliated cells in the terminal bronchiole of the rat.

The administration of 4-ipomeanol, [0, 10 (LD), and 25 (HD) mg/kg, ip], to rats resulted in dose-dependent degeneration and necrosis of the nonciliated (Clara) and ciliated epithelial cells of the terminal bronchioles. More extensive necrosis of the terminal bronchiolar epithelium, with exposure of the basement membrane, was produced in the HD group. Repair of the terminal bronchiolar epithelium was complete within 10 days. Alveolar clearance of 51Cr labeled polystyrene latex microspheres was analyzed through 40 days postinstillation by nonlinear regression for a double exponential model. Alveolar clearance during phase 1 (Days 2 to 6) was delayed and significantly decreased in both the LD and HD groups. Alveolar clearance during phase 2 (Days 10 to 40) was significantly decreased only in the HD group. The decreased alveolar clearance in HD subjects was long term and did not correlate with the return of morphologically normal appearing Clara and ciliated cell structure.