A. Yagminas

Toxicity of 2,2′,4,4′,5,5′-Hexachlorobiphenyl in Rats: Effects Following 90-Day Oral Exposure

The subchronic toxicity of 2,2′,4,4′,5,5′‐hexachlorobiphenyl (PCB 153) was investigated in rats after 13 weeks of dietary exposure. Groups of 10 male and 10 female rats were administered PCB 153 in their diet at levels of 0.05, 0.50, 5.0 or 50 ppm for 13 weeks. The control groups received the diet containing 4% corn oil. Growth rate and dietary consumption were not affected by treatment. Clinical signs of toxicity were not observed. Enlarged, fatty liver was observed in treated animals at necropsy, but most were confined to the two highest dose groups. Increased hepatic microsomal ethoxyresorufin‐O‐deethylase, aminopyrine‐N‐demethylase and aniline hydroxylase activities occurred in high‐dose groups of both sexes, with increased ethoxyresorufin‐O‐deethylase activity being observed starting at 0.05 ppm in females and at 0.5 ppm in males. Treatment‐related reduction in hepatic and pulmonary vitamin A was seen in the highest dose group of both sexes. Changes in brain biogenic amines and intermediate products were observed mainly in females; these included decreased dopamine and 5‐hydroxytryptamine concentrations in the frontal cortex region, and dihydroxyphenylacetic acid in the caudate nucleus region at 5.0 and 50 ppm. Female rats appeared to be more sensitive to the neurotoxic effects of PCB 153 than males. Dose‐dependent histological changes were observed in the thyroid and liver of rats of both sexes and significant changes occurred at 5.0 and 50 ppm. Based on these data, the no‐observable‐adverse‐effect level (NOAEL) of PCB 153 was judged to be 0.5 ppm in the diet or 34 μg kg−1 body wt. day−1.

Subchronic toxicity of PCB 105 (2,3,3′,4,4′-pentachlorobiphenyl) in rats

The toxicity of 2,3,3′,4,4′‐pentachlorobiphenyl (PCB 105) was investigated in Sprague‐Dawley rats following dietary exposure to this substance at levels of 0, 0.05, 0.5, 5 or 50 ppm for 13 weeks. Growth rate and food consumption were not affected and no clinical signs of toxicity were observed. Increased incidences of enlarged, fatty liver and decreased thymic weight were observed in the highest‐dose groups of both genders; these groups also had elevated hepatic microsomal ethoxyresorufin deethylase activity and uroporphyrin. Significant increases in serum cholesterol and hepatic pentoxyresorufin dealkylase activity were observed in the highest‐dose males and two highest‐dose females. By contrast, liver UDP‐glucuronosyl transferase activity was elevated in the two highest‐dose males and the highest‐dose females. Urinary ascorbic acid excretion was increased in the highest‐dose males. While the amount of vitamin A was decreased dose‐dependently, starting at 0.5 ppm in the liver of both sexes and in the lung of the females, the level in the kidney of the highest‐dose group was increased. Administration of PCB 105 resulted in decreased dopamine in the caudate nucleus region of the brain in males and homovanillic acid in caudate nucleus and nucleus accumbens of females. Increased 5‐hydroxytryptamine and 5‐hydroxyindoleacetic acid were observed in the substantia nigra region of both sexes, with most of the increases being seen in highest‐dose females. Anemia, characterized by decreased hemoglobin, hematocrit and red cell indices, occurred in the highest‐dose group, as did eosinophilia. Treatment with PCB 105 caused dose‐dependent histopathological changes in the liver and thyroid. Thymic changes were observed in the highest‐dose males and two highest‐dose females. Tissue residue data showed a dose‐dependent accumulation of this congener in fat, liver and spleen, kidney and brain. Based on these data the no‐observable‐effect level of PCB 105 was judged to be 0.05 ppm or 3.9 μg kg−1 body wt. day−1 in males and 4.2 μg kg−1 body wt. day−1 in females.

Effects of postnatal exposure to a mixture of polychlorinated biphenyls, p,p'-dichlorodiphenyltrichloroethane, and p-p'-dichlorodiphenyldichloroethene in prepubertal and adult female Sprague-Dawley rats.

The postnatal period is a critical phase of development and a time during which humans are exposed to higher levels of persistent organic pollutants (POPs), than during subsequent periods of life. There is a paucity of information describing effects of postnatal exposure to environmentally relevant mixtures of POPs, such as polychlorinated biphenyls (PCBs), p,p′-dichlorodiphenyltrichloroethane (DDT), and p,p′-dichlorodiphenyldichloroethene (DDE). To provide data useful for the risk assessment of postnatal exposure to POPs, mixtures containing 19 PCBs, DDT, and DDE were prepared according to their concentrations previously measured in the milk of Canadian women, and dose-response effects were tested on the proliferation of MCF7-E3 cells in vitro, and in vivo experiments. Female neonates were exposed by gavage at postnatal days (PNDs) 1, 5, 10, 15, and 20 with dosages equivalent to 10, 100, and 1000 times the estimated human exposure level over the first 24 days of life. The MCF7-E3 cells showed a 227% increase in the AlamarBlue proliferation index, suggesting estrogen-like properties of the mixture, but this was not confirmed in vivo, given the absence of uterotrophic effects at PND21. An increase (511%) in hepatic ethoxyresorufin-o-deethylase activity at the dose 100× was the most sensitive endpoint among those measured at PND21 (organ weight, mammary gland and ovarian morphometry, hepatic enzyme inductions, serum thyroxine and pituitary hormones). In liver samples from older female rats (previously involved in a mammary tumor study [Desaulniers et al., Toxicol. Sci. 75:468–480, 2001]), hepatic metabolism of 14C-estradiol-17β (E2) at PND55 to PND62 was significantly higher in the 1000× compared to the control group, but hepatic detoxification enzyme activities had already returned to control values. The production of hepatic 2-hydroxy-E2 decreased, whereas that of estrone increased with age. In conclusion, the smallest dose of the mixture to induce significant effects was 100×, and mixture-induced changes in the hepatic metabolism of estrogens might be a sensitive indicator of persistent effects.

Behavioral and thyroid effects of in utero and lactational exposure of Sprague–Dawley rats to the polybrominated diphenyl ether mixture DE71

Exposure of rodents during gestation and lactation to polybrominated diphenyl ethers (PBDEs) has been reported to disrupt neurobehavioral function in offspring, as well as to disrupt thyroid function. To assess this we evaluated development and behavior after gestational and lactational exposure to the technical PBDE mixture DE71. Pregnant Sprague-Dawley rats were exposed to 0, 0.3, 3.0 or 30 mg/kg/day of DE71 from gestation day 1 to postnatal day (PND) 21 and were assessed on a wide range of behavioral functions from early postnatal period until old age (PND 450). DE71 exposure decreased thyroid hormone levels (T3 and T4) in mothers and offspring with offspring being more sensitive that mothers. Developmental landmarks, neuromotor function, anxiety, learning and memory were not affected by DE71 at any age. DE71 produced small changes in motor activity rearing only at PND 110 but not at any other age and no other activity measure was altered by DE71. Cholinergic sensitivity measured by nicotine-stimulated motor activity was not affected by perinatal DE71 exposure. Acoustic startle responses were potentiated by DE71 at PND 90 indicating delayed effects on sensory reactivity. Habituation was measured in motor activity tests at five ages but was not altered by DE71 at any age. Habituation measured in startle tests was also not affected by exposure to DE71. For thyroid hormone levels at PND 21, the lowest adverse effect level was 3.0 mg/kg. Few behavioral effects were observed and the lowest adverse effect level was 30 mg/kg. Our results confirm that DE71 produces transient effects on thyroid hormone levels but does not result in learning or motor impairment and does not alter non-associative learning (habituation).

Effects of dibromoacetonitrile on rats following 13-week drinking water exposure

The subchronic toxicity of dibromoacetonitrile (DBAN), a disinfection by-product in drinking water, was studied in the rat. Male (180+/-18 g) and female (152+/-9 g) Sprague-Dawley rats (10 animals per group) were fed DBAN in organic-free distilled water at concentrations of 0.1, 1, 10 and 100 ppm for 13 weeks. Control rats received organic-free distilled water only. Water intakes in the highest dose males and females were reduced by 25 and 32% as compared to the controls, respectively (P<0.05), with no significant reductions in food consumption and body weight gain. The organ to body weight ratio was significantly increased in the highest-dose males and females for kidneys but not for the brain, liver, spleen, thymus and testicles. In the males, decreases were detected in serum uric acid levels at 1 and 100 ppm, and in urinary uric acid at 10 and 100 ppm. Decreased serum protein was detected in the highest-dose males and decreased serum LDH was found in the highest-dose females. Both the white blood cell and lymphocyte counts were significantly elevated in the highest-dose females. A significant increase in hepatic catalase activity was observed only in males starting at 1 ppm, and increased palmitoyl-CoA oxidase (PCO) activity was found in males and females of the highest dose group. In the males, decreased thiobarbituric acid reactive substance (TBARS) level was detected in the liver at 1.0 and 100 ppm groups, while increased TBARS was found in the serum at 100 ppm DBAN. No treatment-related changes were detected in the activities of hepatic benzyloxyresorufin O-dealkylase (BROD), pentoxyresorufin O-dealkylase (PROD) and ethoxresorufin O-deethylase (EROD), and in hepatic UDP-glucuronosyltransferase (UDPGT) and glutathione S-transferases (GST). Although DBAN is a potent inhibitor of hepatic aldehyde dehydrogenase (ALDH) and GST in vitro, there was no evidence of suppression of these enzymes in the treated animals. Mild histological changes were detected in animals receiving the highest dose, consisting of collapsed angularity, increased epithelial height in the thyroid of both sexes, and cytoplasmic vacuolation and nuclear vesiculation in the thyroid of females, increased myeloid to erythroid ratio in the bone marrow of both sexes, and cytoplasmic inclusions in the proximal tubules of male kidneys. In summary, treatment effects occurred predominantly at 100 ppm and included in both sexes: increased kidney weights, histological changes in the thyroid and bone marrow, and increased peroxisomal enzyme activities; and in males: decreased serum and urinary uric acid levels, and indication of oxidative stress. The no-observed-adverse-effect level (NOAEL) was therefore judged to be 10 ppm, equivalent to 1.11 and 1.21 mg/kg/day in the males and females, respectively.

90-Day Repeated Inhalation Exposure of Surfactant Protein-C/Tumor Necrosis Factor-α (SP-C/TNF-α) Transgenic Mice to Air Pollutants

Tumor necrosis factor (TNF)-α, a cytokine present in inflammed lungs, is known to mediate some of the adverse effects of ozone and inhaled particles. The authors evaluated transgenic mice with constitutive pulmonary expression of TNF-αunder transcriptional regulation of the surfactant protein-C promoter as an animal model of biological susceptibility to air pollutants. To simulate a repeated, episodic exposure to air pollutants, wild-type and TNF mice inhaled air or a mixture of ozone (0.4 ppm) and urban particles (EHC-93, 4.8 mg/m3) for 4 h, once per week, for 12 consecutive weeks and were sacrificed 20 h after last exposure. TNF mice exhibited chronic lung inflammation with septal thickening, alveolar enlargement, and elevated protein and cellularity in bronchoalveolar lavage fluid (genotype main effect, p <.001). Repeated exposure to pollutants did not result in measurable inflammatory changes in wild-type mice and did not exacerbate the inflammation in TNF mice. The pollutants decreased recovery of alveolar macrophages in lavage fluid of both wild-type and TNF mice (exposure main effect, p < .001). Exacerbation of the rate of protein nitration reactions specifically in the lungs of TNF mice was revealed by the high ratio of 3-nitrotyrosine to L-DOPA after exposure to the air pollutants (Genotype × Exposure factor interaction, p = .014). Serum creatine kinase-MM isoform increased in TNF mice exposed to pollutants (Genotype × Exposure factor interaction, p = .043). The marked pollutant-related nitration in the lungs of the TNF mice reveals basic differences in free radical generation and scavenging in the inflamed lungs in response to pollutants. Furthermore, elevation of circulating creatine kinase-MM isoform specifically in TNF mice exposed to pollutants suggests systemic adverse impacts from lung inflamma-tory mediators, possibly on muscles and the cardiovascular system.

A Study of the Acid Lability and Acute Toxicity of Dimethoxymethane in Rats

The pH stability and acute oral toxicity of dimethoxymethane (DMM) in rats were studied. In in vitro studies, DMM was found to be stable in artificial gastric juice at pH 2.5 and above but readily hydrolyzed at pH 2 and below, with 1 mole of DMM producing approximately 1 mole of formaldehyde and 2 moles of methanol. DMM incubated in sealed, excised stomachs appeared to equilibrate with the external incubation fluid over a 24-hour period without significant hydrolysis. In the toxicity study, three groups of female rats (n = 5) were administered, by gavage, phosphate-buffered saline (control), DMM (31 mmoles/kg body weight [bw]/day), or methanol (MeOH) (30 mmoles/kg bw/day), respectively, for 3 consecutive days and then sacrificed on day 5. Transient ataxia lasting about 30 minutes was observed in animals following DMM administration. Increased urine volume was observed in the DMM group at the end of days 4 and 5. Markedly elevated level of formic acid was present in the urine of the MeOH group at days 1, 2, and 3, but not in the DMM group. Urinary ascorbic acid levels were increased in the DMM group on days 1, 2 and 3, and returned to baseline level at days 4 and 5. Microscopic examination detected no abnormal histological changes in the liver and kidneys of MeOH-or DMM-treated animals. No treatment effects were observed on the following endpoints: organ weights (liver, heart, thymus, kidneys), serum corticosterone, serum chemistry profile, hematology, hepatic phase 1 and phase 2 mixed-function oxidase activities, hepatic and serum thiobarbituric acid, hepatic glutathione, urinary protein, and urinary N-acetylglucosaminidase activity. These observations suggested that hydrolysis of DMM in the stomach required a gastric acidity below pH 2.5. At higher pH, intact DMM readily penetrated the stomach and therefore likely reached the circulation and other body tissues. There was no significant hydrolysis of orally administered DMM, and the acute effects were limited to transient ataxia and biochemical responses in the liver.

Activity of erythrocyte δ-aminolevulinic acid dehydratase in the female cynomolgus monkey (Macaca fascicularis): kinetic analysis in control and lead-exposed animals

Kinetic analysis of erythrocyte delta-aminolevulinic acid dehydrase (delta-ALAD) from female cynomolgus monkeys revealed differences in pH optimum and Michaelis constants according to their exposure to lead. In vitro incubation of delta-ALAD with 5 mM dithiothreitol (DTT) or 100-200 microM zinc resulted in an enhanced enzyme activity being expressed. These effects were additive. Activation with DTT or zinc resulted in the abolition of pH differences between control and exposed animals and revealed an increased quantity of enzyme in exposed animals. delta-ALAD in control monkeys was observed to be very sensitive to inhibition by lead in vitro with an apparent inhibition constant (Ki) of 0.12 microM. The effect of lead on monkey delta-ALAD enzyme kinetics is similar to that seen with human samples and thus is a useful model for measuring biological response to lead exposure.