James E. Andrews

Chlorpyrifos elicits mitotic abnormalities and apoptosis in neuroepithelium of cultured rat embryos

Chlorpyrifos is used increasingly as a pesticide in place of more toxic alternatives such as parathion. Although chlorpyrifos is not a potent dysmorphogen, recent reports that fetal or infant exposures may exceed acceptable limits have raised concern about the potentially more subtle effects on brain development. In the current study, whole rat embryo culture was used to study the effects of chlorpyrifos at the neural tube stage of development. On embryonic day 9.5, embryos were exposed to 0.5, 5, or 50 micrograms/ml of chlorpyrifos. After 48 hr (embryonic day 11.5), embryos were examined for dysmorphogenesis and were then processed for light microscopic examination of the neuroepithelium. Examination of 1-micron-thick toluidine blue-stained sections of the forebrain and hindbrain region revealed reduced and altered mitotic figures, with dispersion and disorientation of the mitotic layer. In addition, cytotoxicity was evidenced by cytoplasmic vacuolation, enlargement of intercellular spaces, and the presence of a significant number of apoptotic cells. These alterations were evident even at the lowest concentration of chlorpyrifos, which produced no dysmorphogenesis. The effects were intensified at higher concentrations, which were just at the threshold for dysmorphogenesis; the neuroepithelial abnormalities, however, were still present in embryos that were not dysmorphogenic. Our results in rat embryo culture support the idea that chlorpyrifos specifically targets brain development at low concentrations, indicating the need to reevaluate the safety of this compound for exposure in vivo.

Polychlorinated biphenyl (aroclor 1254) induced changes in femur morphometry calcium metabolism and nephrotoxicity

Experiments were performed to investigate the effects of polychlorinated biphenyl (PCB) on calcium metabolism, femur morphometry, and nephrotoxicity. Male Fischer 344 rats were dosed daily intragastrically (ig) for 5, 10 or 15 weeks with 0, 0.1, 1, 10 or 25 mg PCB/kg body weight. After 5, 10 and 15 weeks, liver weight and liver-to-body weight ratio were increased at the 2 higher dose levels. Overt toxicity was observed at the highest dose level after 10 and 15 weeks as evidenced by significantly smaller body weight. Urinary alkaline phosphatase and lactate dehydrogenase activities were elevated at 5, 10 and 15 weeks of PCB exposure and the kidney-to-body weight ratios were elevated at the 10 and 25 mg/kg dose levels after 10 and 15 weeks of exposure indicating nephrotoxicity. Hypercalcemia was present at the highest dose level after 5 and 10 weeks of exposure but serum calcium concentration was normal at 15 weeks. Serum triglycerides were significantly elevated after 5 weeks of exposure but were significantly decreased after 10 and 15 weeks of PCB exposure. Serum cholesterol was significantly elevated at the 2 higher dose levels at all 3 periods. Femur density was increased at the 10 mg/kg dose level after 5 weeks, at all dose levels after 10 weeks and at all dose levels which did not demonstrate overt toxicity after 15 weeks of PCB exposure. Cross-sectional, medullary and cortical areas of the midpoint of the femur were significantly decreased at the higher dose levels after 10 and 15 weeks of exposure. The percent medullary area though was significantly decreased after 10 and 15 weeks of PCB exposure indicating that not only was there a decrease in medullary size but a decrease relative to the cortical bone area. These changes in bone morphometry resulted in a significantly weaker bone after 15 weeks of exposure at the highest dose level. These results demonstrate that PCB exposure effects calcium metabolism and bone morphometry and is nephrotoxic.

A Perinatal Study of Toluene in CD-1 Mice

Toluene administered by inhalation at 400 ppm to CD-1 mice from Days 6 to 16 of gestation was teratogenic but not fetotoxic resulting in a significant shift in the fetal rib profile. At the lower concentration of 200 ppm, there was an increase in dilated renal pelves which might reflect desynchronization of maturation with respect to development and growth. No other effects were noted at the 200-ppm concentration. At 400 ppm, toluene also produced an increased body weight in the neonates on Day 1 postpartum following in utero exposure. Activity of lactic dehydrogenase (LDH) was significantly increased in the brains of dams exposed to 400 ppm during gestation while nonpregnant adult mice studied concurrently had significant increased activities of LDH in the liver and kidneys of the 400-ppm group. The only change in the isozyme profiles was in the kidneys of the nonpregnant adult mice in which a slight decrease in LDH-2 was observed. No other changes were noted in the dams or pups.

The effects of lindane and linuron on calcium metabolism, bone morphometry and the kidney in rats.

Experiments were performed to investigate the effects of lindane and linuron on calcium metabolism, femur morphometry and nephrotoxicity. Long-Evans hooded rats were dosed daily for 10 weeks with 0, 10 or 20 mg lindane or 10, 20 or 40 mg linuron/kg body weight beginning at weaning. Lindane significantly decreased urinary calcium concentration, serum alkaline phosphatase concentration and the cross-sectional medullary area of the bone. Lindane was nephrotoxic at both dose levels as demonstrated by elevated kidney weights, kidney-to-body-weight ratios, urinary LDH, tubule regeneration and hyaline droplet degeneration. Linuron significantly reduced medullary cross-sectional area at the 2 higher dose levels and decreased the total femur cross-sectional area at the highest dose level in the absence of effects on calcium excretion. Femur density and strength were also significantly reduced at the highest dose level of linuron. Neither compound affected the serum concentrations of parathyroid hormone or 1,25-dihydroxy Vitamin D-3. Both linuron and lindane exposure significantly increased serum cholesterol concentrations and reduced serum triglyceride concentrations. Both compounds affected calcium metabolism and/or bone morphometry but possibly by different mechanisms since the effects were not the same.

Hexachlorobenzene-Induced Hyperparathyroidism and Osteosclerosis in Rats,

Hexachlorobenzene (HCB) exposure has been shown to alter the normal concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D3 in rats and to result in osteoporosis in humans. Experiments were undertaken to investigate the effects of HCB on the homeostatic mechanism of calcium metabolism and to determine its effect on bone in rats. Fischer 344 rats were dosed 5 days/week for 5, 10, or 15 weeks with 0, 0.1, 10.0, or 25.0 mg HCB/kg body wt. Body weight was not affected by any of the exposure conditions. Liver weight was significantly elevated above control values at the two higher dose levels at all three time periods. Kidney weight and kidney-to-body weight ratio were significantly elevated at the highest dose level after 10 weeks and at the two higher dose levels after 15 weeks of exposure. Serum alkaline phosphatase was significantly decreased at the two higher dose levels after both 10 and 15 weeks of exposure. 1,25-Dihydroxyvitamin D3 was measured in the 5-week exposure group only and was significantly elevated in the three higher dose levels. After 5 and 15 weeks of HCB exposure, parathyroid hormone concentration was significantly elevated at the two higher dose levels at both time periods. Wet femur density was significantly increased at the two higher dose levels of HCB after 10 weeks of exposure and the three higher dose levels after 15 weeks of exposure. Dry femur density was also increased in the cases where wet femur density was increased. However, femur weight was not affected at any dose level. The results from this study indicate that HCB induces hyperparathyroidism in rats, as demonstrated by increased serum parathyroid hormone levels and osteosclerosis of the femur.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric analysis of osteosclerotic bone resulting from hexachlorobenzene exposure

Hexachlorobenzene (HCB) exposure has been shown to induce hyperparathyroidism and osteosclerosis in rats. Experiments were undertaken to investigate the effects of HCB-induced hyperparathyroidism and osteosclerosis on femur morphometry as well as femur breaking strength. Fischer 344 rats were dosed 5 d/wk for 15 wk with 0, 0.1, 1, 10, or 25 mg HCB/kg body weight. Hyperparathyroidism was produced in the two higher dose groups as reported previously (Andrews et al., 1989). Femur weight was significantly increased in the rats receiving 0.1, 1, and 25 mg HCB/kg body weight, whereas density was increased significantly at 1, 10, and 25 mg HCB/kg dose levels. Bone strength was also significantly increased at the three higher dose levels. Cross-sectional area of the midpoint of the femur was significantly increased at the 1 mg/kg HCB dose level. Cortical area and the proportion of the total area of the bone that the cortex occupied were significantly increased at the three higher dose levels. Medullary cavity area was significantly increased at the 0.1 mg/kg dose level but significantly decreased at the 2 higher dose levels of HCB. The right femur was significantly predominant to the left femur in weight, volume, and density through all dosing regimens. HCB exposure significantly altered bone morphometry and strength characteristics in the Fischer 344 rat.

Teratogenic evaluation and fetal deposition of hexabromobenzene (HBB) And hexafluorobenzene (HFB) in CD‐1 mice

HBB (hexabromobenzene) and HFB (hexafluorobenzene) were tested for their teratogenic potential in CD-1 mice. HBB and HFB were administered to pregnant mice from the 6th to the 16th day of gestation by gastric intubation. Neither HBB nor HFB were teratogenic or fetotoxic at doses up to 98.6 mg HBB/kg and 65.3 mg HFB/kg. No maternal toxicity was noted. HBB concentration in the fetuses indicated little, if any accumulation. No HFB was detected in the fetal or maternal tissues 24 hours after the last dose.Abstract HBB (hexabromobenzene) and HFB (hexafluorobenzene) were tested for their teratogenic potential in CD‐1 mice. HBB and HFB were administered to pregnant mice from the 6th to the 16th day of gestation by gastric intubation. Neither HBB nor HFB were teratogenic or fetotoxic at doses up to 98.6 mg HBB/kg and 65.3 mg HFB/kg. No maternal toxicity was noted. HBB concentration in the fetuses indicated little, if any, accumulation. No HFB was deteced in the fetal or maternal tissues 24 hours after the last dose.

Impairment of calcium homeostasis by hexachlorobenzene (HCB) exposure in Fischer 344 rats.

Human exposure to hexachlorobenzene (HCB) has resulted in demineralization of bone and development of osteoporosis. Experiments were undertaken to investigate the effects of HCB on the homeostatic mechanism of calcium metabolism. Fischer 344 rats were dosed with 0, 0.1, 1.0, 10.0, or 25.0 mg HCB/kg body weight 5 d/wk for 5 wk while being fed normal rat diet or vitamin D3-deficient diet. Rats receiving the normal diet had a dose-related decrease in body weight gain and increased liver weight when compared to their controls. Serum cholesterol, alanine aminotransferase (ALT), 1, 25-dihydroxy-vitamin D3 [1,25-(OH)2D3], and parathyroid hormone (PTH) were significantly elevated when compared to control values. In the vitamin D3-deficient diet group, there was a dose related increase in liver weight, liver-to-body weight ratio and kidney-to-body weight ratio. Serum cholesterol and 1,25-(OH)2D3 were significantly elevated. Urinary calcium decreased significantly with increasing HCB dosage, indicating conservation of calcium. The data from this study indicate that HCB does affect calcium metabolism by altering the concentrations of two primary controlling factors in calcium homeostasis.

Stereoselective dysmorphogenicity of the enantiomers of the valproic acid analogue 2-N-propyl-4-pentynoic acid (4-yn-VPA): Cross-species evaluation in whole embryo culture

We previously reported the in vitro differential stereoselective dysmorphogenic potential of the R(+) and S(-) enantiomers of 2n-propyl-4-pentynoic acid (4-yn-VPA) in mice. To determine whether this stereoselectivity is species specific, we evaluated the dysmorphogenic potential of these isomers as well as valproic acid (VPA) to gestational day 9 rat embryos using whole embryo culture (WEC). Aqueous solutions of the sodium salts of R-4-yn-VPA, S-4-yn-VPA, 50%R/ 50%S-4-yn-VPA or VPA were added to the culture medium to give 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/L and embryos were evaluated 48 hr later. The S-4-yn-VPA enantiomer gave clear concentration-dependent dysmorphology as well as effects on developmental score, somite number, crown rump length, and head length. Effects on rotation and defects of the neural tube, somites and heart were observed. Embryolethality was observed only at 1.2 mmol/L concentration. The R-4-yn-VPA enantiomer was neither embryo toxic nor dysmorphogenic at any concentration. VPA significantly reduced all parameters and was dysmorphogenic at the highest concentration but was not embryo lethal. The 50/50 mixture of R- and S-isomers appeared to elicit a degree of embryolethality and dysmorphology similar to VPA. The potency order for the four chemicals was S(-) > S(-)/R(+) = VPA > > > R(+), comparable to that observed in mice by either in vivo or in vitro exposure. These data demonstrate that the stereoselective dysmorphology for these enantiomers can be observed across species and is not related to maternal metabolism.

Assessment of teratogenic potential of trichlorfon in mice and rats

Trichlorfon was evaluated for its teratogenic potential in the CD-1 mouse at doses of 200, 300 or 400 mg/kg/day administered by gavage on days 7-16 of gestation. In the CD-1 mouse, TCF was teratogenic, fetotoxic and lethal at the two highest dose levels which were also maternally lethal. At the lowest dose level which was not maternally lethal, there was a significant decrease in the number of calcified centers in the forepaws and hindpaws indicating fetotoxicity and a delay in maturation. TCF was administered at doses of 50, 100 or 200 mg/kg/day to CD rats by gavage on gestational days 7-19 (study I) or 8-20 (study II). In the CD rat in body study I and II, the highest dose level was maternally lethal. In study I, TCF was teratogenic with a shift in rib profile. In study II, TCF was teratogenic with an increased incidence in malformations of the urinary system. Additionally, TCF was fetotoxic with reduced ossification of the skulls at the lowest and highest dose levels.