Behavioral toxicity in the offspring of rats following maternal exposure to dichloromethane

Rats divided in four treatment groups were exposed to dichloromethane (DCM) (4500 ppm) or filtered air before and/or during gestation in order to assess the occurrence and extent of toxic effects on developing offspring. The progeny of dams exposed to DCM either prior to and/or during gestation exhibited altered rates of behavioral habituation to novel environments. No simple relationship between exposure period and behavioral outcome was observed. Each of the treatment groups showed effects as a function of age at testing and the behavioral task used. Treatment effects were detectable in offspring as early as 10 days of age and were still demonstrable in 150-day-old male rats. Treatment effects were observed in rats of both sexes in preweaning tests but were not seen in adult female rats. No effects of subacute DCM exposure were evident in growth rate, long-term food and water consumption, wheel running activity, or avoidance learning. This study, which should be viewed as preliminary, is cf interest since altered rates of habituation to novel environments were observed in the absence of overt maternal toxicity, or teratogenicity. The effects cannot be definitely attributed to a direct effect of DCM since elevated maternal carboxyhemoglobin (COHb)- or DCM-induced changes in maternal-litter interactions could have been contributing factors. The findings do suggest that the functional development of progency of DCM-exposed dams should be further investigated.     

Comparative toxicity of netilmicin and tobramycin in dogs

Beagle dogs (four/group) were dosed intramuscularly with netilmicin or tobramycin once daily at doses of 20, 40, or 80 mg/kg. The dogs were dosed for 30 days or until death or sacrifice because of poor condition. Weekly otological examinations (response to Galton whistle) were conducted to evaluate effects on hearing. Hematologic, serum chemical and urinalysis data were collected weekly. Kidneys and cochleas were examined histologically. Dogs given 40 and 80 mg/kg of tobramycin became azotemic and died or were sacrificed in a moribund condition 9 to 22 days after dosing was initiated. Serum urea nitrogen and creatinine levels increased in $\text{2}\text{4}$ dogs given tobramycin at 20 mg/kg. No clinical signs of toxicity or unusual serum chemical changes occurred in the dogs dosed with netilmicin. Hearing did not appear to be affected by either compound. Microscopic examination of kidneys revealed tubular epithelial necrosis ranging from mild (netilmicin) to severe (tobramycin). Steady-state serum drug concentrations were established at all dose levels for netilmicin. In contrast, serum concentrations of tobramycin increased at all dose levels apparently as a result of nephropathy. Under the conditions of this study, netilmicin was clearly less toxic than tobramycin.     

Influence of concentration and rate of intravenous administration on the toxicity of cyclohexanone in beagle dogs

The importance of concentration and rate of intravenous administration in affecting the toxicity of cyclohexanone was investigated. Four groups (I–IV) comprised of three male dogs each received a dosage of 284 mg/kg/day for 18–21 days as follows: 6% ($\text{v}\text{v}$) solution at 75 (I) or 5 ml/min (II); or 0.75% solution at 75 (III) or 5 ml/min (IV). Signs of toxicity observed included vocalization, lacrimation, scleral vasodilation, mydriasis, salivation, urination, defecation, restlessness, stupor, ataxia, occasional convulsive movements, hyperpnea, and/or dyspnea. Also, regimen I produced a metabolic acidosis with an apparent respiratory component. The severity of the responses correlated well to the maximal plasma concentrations of cyclohexanone attained, which ranged from 80 to 320 μg/ml, and to the conditions of administration as follows: I > II > III > IV. This distinction became even more apparent with repeated administrations and affected the prognosis for recovery. The concentration of cyclohexanone given was more critical than the rate of injection in producing localized tissue inflammation at injection sites, hemolysis, and secondary responses, e.g., bone marrow hyperplasia and extramedullary hematopoiesis. The rate of administration was also important since, at either concentration of cyclohexanone employed, the group receiving the compound at a faster rate had a greater response. Thus, the influence of iv administration factors on cyclohexanone toxicity was great, showing the importance of taking such factors into account in any safety evaluation study.     

Stimulation of DNA synthesis and pyrimidine deoxyribonucleoside transport systems in mouse glioma and mouse neuroblastoma cells by inorganic mercury

Biochemical studies on effects of methylmercury and inorganic mercury on mouse glioma and neuroblastoma cells were carried out. Inorganic mercury accelerated DNA synthesis of mouse glioma cells at 10^?6 to 10^?5m specifically in the middle log phase of the growth. Inorganic mercury facilitated the thymidine and deoxycytidine transports within a narrow range of concentration (1 to 2 × 10^?5m), but no stimulation of transport was observed in the cases of purine deoxyribonucleosides. Inorganic mercury markedly inhibited both DNA synthesis and the transport systems of the precursors above 5 × 10^?5m. On the other hand, methylmercury showed hardly any accelerating effects and markedly inhibited both the synthesis of DNA and the precursors' transport above 5 × 10^?5m. In the case of mouse neuroblastoma cells, inorganic mercury stimulated DNA synthesis at 1 to 5 × 10^?6m and also thymidine transport system at 1 to 2 × 10^?5m.     

Effects of superoxide dismutase and catalase on central nervous system toxicity of hyperbaric oxygen

The effects of exogenous superoxide dismutase (SOD) (20 mg/kg) and catalase (3.0 mg/kg) and the combination of SOD + catalase upon the time to convulsions following 100% oxygen at 3 and 6 atm have been studied in mice. SOD alone produced no change in the time to convulsions, catalase produced a prolongation of approximately 50% of the time to convulsions observed in the untreated mice, and SOD when administered with the catalase interfered with the catalase response. Graded doses of catalase produced a graded prolongation of time to convulsions, demonstrating a dose-response relationship. These findings are compatible with previous investigations showing an increase in brain H₂O₂ with exposure to hyperbaric oxygen, which may infer that this substance plays a part in the CNS toxicity observed.     

Alteration of vascular permeability due to cytochalasin E

Cytochalasin E increased the vascular permeability of the rat. It induced leakage through the endothelial gaps of the venules and capillaries without affecting the arterioles.     

Biochemical and morphological assessments of bleomycin pulmonary toxicity in rats

Low doses (5 units) or high doses (15 units) of bleomycin were administered ip twice weekly to separate groups of male rats. A progressive dose-dependent reduction in body weight gain with a concomitant increase in wet lung weight to body weight ratio was observed over 6 weeks of treatments. Light microscopic confirmation of perivascular edema suggested that the increase in wet lung weight resulted from fluid accumulation. Although histological evidence of mild fibrosis was also present following low-dose treatment, only a slight increase in collagen content was observed biochemically. In vivo elevation of prolyl hydroxylase activity was observed in lungs of animals treated with low-dose bleomycin for 6 weeks. High-dose bleomycin regimen did not accelerate fibrosis, but rather inhibited the prolyl hydroxylase activity. In vitro, this enzyme activity was also stimulated at 1–10 μm concentration, but was inhibited at 0.1 mm. Lung angiotensin-converting enzyme activity was examined as an indication of damage to endothelial cells. This activity was unchanged after low-dose bleomycin treatments until 6 weeks, at which time a 59% decrease in activity was observed. High-dose bleomycin treatment produced a biphasic response in angiotensin-converting enzyme activity, with an initial 32% stimulation after 1.5 weeks of treatment followed by a 41% inhibition after 3 weeks. Kinetic analysis of the in vitro interaction of purified porcine plasma angiotensin-converting enzyme with bleomycin indicated that bleomycin acts as a competitive inhibitor with a K_i of 3.7 μm. Stimulation of angiotensin-converting enzyme activity following acute high-dose bleomycin administration could be due to the initial endothelial cell repair, while the inhibition at later stages may indicate extensive destruction of endothelial surface upon repeated bleomycin administration. Direct inhibition of the enzyme by the chelating effect of bleomycin may also be a possibility.     

Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes

The toxicity of cadmium may be due to alteration in membrane structure which may be caused by peroxidation of the composite lipids. Isolated hepatocytes provide a suitable system to examine the role of lipid peroxidation in a toxic response at the cellular level. Therefore, isolated rat hepatocytes were incubated with varying cadmium concentrations (50–400 μm) for up to 75 min. An increase in lipid peroxidation due to cadmium was observed. The integrity of the cell membrane, as measured by loss of intracellular potassium ion and leakage of aspartate aminotransferase, was adversely affected in the presence of cadmium. The lactate to pyruvate ratio of hepatocyte suspensions was increased upon incubation with cadmium. Several chelating compounds were found to reduce intracellular accumulation of cadmium, cellular toxicity and lipid peroxidation. However, amelioration of toxicity was not consistently associated with inhibition of the lipid peroxidation response. The antioxidant compounds, sodium diethyldithiocarbamate and N,N′-diphenyl-p-phenylene-diamine were found to inhibit the lipid peroxidation attributable to cadmium, but did not have any consistent protective effect against loss of intracellular potassium ion. The results of this study show that the toxicity induced by cadmium in isolated rat hepatocytes can be dissociated from the concurrently observed lipid peroxidation, which indicates that the toxic response is not caused by the lipid peroxidation.     

Postnatal development of constitutive forms of cytochrome P-450 in liver microsomes of male and female rats

In a previous paper (1), we reported on the purification of constitutive forms of cytochrome P-450, namely P-450-male and P-450-female, from liver microsomes of male and female rats, respectively. Immunochemical examinations of these hemoproteins showed that P-450-male and P-450-female were detectable specifically in respective liver microsomes of adult male and female rats. The synthesis of P-450-male was apparently dependent on testosterone, and that of P-450-female was dependent on estradiol.Thus, in this study we examined the postnatal development of P-450-male and P-450-female. We show herein that P-450-female is primarily synthesized before the occurrence of P-450-male in male rats. This and other results support the view that the synthesis of unknown pre-existing forms of cytochrome P-450 is depressed in association with the appearance of P-450-male and P-450-female during postnatal periods before sexual maturation.     

Comparative neurotoxicity and metabolism of ethyl n-butyl ketone and methyl n-butyl ketone in rats

The six-carbon compounds methyl n-butyl ketone (MnBK) and n-hexane are metabolized by oxidation of their subterminal carbons through a series of steps leading to the production of 2,5-hexanedione (2,5Hxdn). Each oxidative step increases the neurotoxic potential so that 2,5Hxdn is the most active compound in the metabolic series. The most likely seven-carbon compound commercially available that would produce a metabolic analog to 2,5Hxdn is ethyl n-butyl ketone (EBK). A study was conducted to determine the neurotoxicity of EBK and to identify its major metabolites. Rats were exposed to 700 ppm MnBK by inhalation for 11 weeks or EBK for 24 weeks, twice the exposure period needed to produce a severe, clinically evident neuropathy with MnBK. No clinical or neuropathological evidence of neurotoxicity was evident after EBK exposure. These findings appear to be due to the low serum concentrations of 2,5-heptanedione (6.8 ± 4.0 μg/ml) formed from EBK in comparison to the much higher concentrations of 2,5Hxdn (133.2 ± 36.7 μg/ml) formed from MnBK. This argument is strengthened by the fact that in a companion study, 2,5-heptanedione administered to rats 5 days per week by gavage in 1000 and 2000 mg/kg/day doses was found to produce the same type of neurotoxicity as seen with 2,5-Hxdn.     

Effects of chemical agents on the hepatotoxicity and hepatic accumulation of luteoskyrin

Pretreatment of male mice with 3-methylcholanthrene and promethazine prevented the hepatic accumulation and the hepatotoxicity of luteoskyrin, while piperonyl butoxide and cobaltous chloride had no effect on either. Pretreatment with SKF-525 A elevated SGPT activity in male and female mice. Pretreatment with cycloheximide increased hepatic luteoskyrin accumulation in male mice. Dibenamine prevented hepatic luteoskyrin accumulation and hepatotoxicity only when it was injected in two doses, one the day before and the other concurrently with luteoskyrin. The data suggest that biliary excretion of luteoskyrin may be stimulated by an enzyme other than the microsomal drug metabolizing enzymes. 3-Methylcholanthrene, promethazine and dibenamine may accelerate this excretion thus protecting against luteoskyrin-induced hepatotoxicity. Cycloheximide may prevent this excretory process. The microsomal drug metabolizing enzymes may participate in the detoxification processes of the active form of luteoskyrin.     

Toxicologic and acute lethal hazard evaluation of thermal decomposition products of synthetic and natural polymers

Thermal decomposition products from a series of cellular synthetic polymers and several other polymers, synthetic or natural, were investigated. Evaluation of sensory irritating properties, physiological stress, asphyxiation, acute mortality, and histopathological changes due to the thermal decomposition products revealed a very wide range of potency among them. A classification system is proposed for making comparison to standard materials such as wood or fiberglass insulation and an acute lethal hazard system is presented for comparison of materials used for the same function, i.e., insulation.     

The toxicological evaluation of the mycotoxins T-2 and T-2 tetraol using normal human fibroblasts in vitro

Normal human fibroblasts in vitro were used as a system to examine the cellular effects of potentially toxic compounds. In addition to tests for viability, indices of toxicity were included which would demonstrate functional and structural alterations associated with specific subcellular components. Fibroblast cultures derived from human neonatal foreskin were treated with various doses of the mycotoxins T-2 and T-2 tetraol. Methods for demonstrating viability showed T-2 to be 10-fold more toxic than its hydroxylated derivative, T-2 tetraol. Both compounds induced a dose-dependent reduction in protein and scheduled DNA synthesis, as well as an induction of unscheduled DNA synthesis (DNA repair). However, no qualitative alterations in the activity of cytoplasmic, lysosomal or membrane-associated enzymes were observed with T-2 or T-2 tetraol-treated cells. Transmission electron microscopy revealed structural alterations predominantly associated with the nucleus and the endoplasmic reticulum with associated ribosomes. Multiple biochemical parameters with ultrastructural and cytotoxicity studies were therefore effective in demonstrating the mechanisms as well as the degree of toxicity induced by these chemical compounds.     

Toxicity of mercuric chloride in cultures of neurons and nonneuronal cells derived from embryonic chick sympathetic ganglia

The effects of mercuric chloride were studied in vitro using both mixed and highly purified cultures of neurons and nonneuronal (glial) cells. Cultures were prepared from the sympathetic ganglia of 11-day chick embryos and treated with mercuric chloride (0.1 nm to 100 μm) for either 2 or 3 days. Acetyl cholinesterase specific activity, [³H]thymidine incorporation per microgram of protein, and [¹⁴C]leucine incorporation per microgram of protein were quantified, and the following results were obtained. First, all three biochemical measures either were stimulated or were unaffected by exposure of mixed ganglion cell cultures to ≤10 μm mercuric chloride for 2 days. In contrast, exposure to 100 μm mercuric chloride for 2 days depressed incorporation of both leucine and thymidine, but stimulated acetyl cholinesterase specific activity. Second, incorporation of both leucine and thymidine rapidly returned to control levels following removal of the mercuric chloride from the culture medium after 2 days of exposure. In contrast, acetyl cholinesterase specific activity remained elevated. Third, all biochemical measures were either inhibited or unaffected by a 3-day exposure of mixed ganglion cell cultures to any concentration of mercuric chloride. Fourth, sympathetic neurons appeared to be much more sensitive to mercuric chloride than homologous nonneuronal cells. Fifth, most of the stimulatory and inhibitory effects of mercuric chloride appeared to result from direct action of the heavy metal on either the neurons or nonneuronal cells.     

Mutagenicity testing of agent orange components and related chemicals

Components of the herbicide Agent Orange--2,4-dichlorophenoxyacetic acid (2,4,-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and their esters, and the contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)--and related chemicals were tested for mutagenicity using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No mutagenic activity was observed for any of the chemicals tested.     

Embryotoxic,teratogenic, and metabolic effects of ribavirin in mice

To gain comparative perspective on the teratogenic mechanisms of action of drugs that are precursor analogs or antimetabolites of nucleic acids, we employed a new antiviral agent—ribavirin—which is known in other systems to inhibit the biosynthesis of guanine nucleotides. Pregnant ICR mice were injected at 10th–13th days of gestation (stages 14–20) with a single ip dose of ribavirin in the range of 10–200 mg/kg. All dosages in excess of 25 mg/kg were teratogenic. The optimal teratogenic dose varied with the stage of development, being higher at advanced stages of development. Depending on the dose and stage of treatment, virtually all parts of the skeleton including the craniofacial and limb bones were susceptible to ribavirin. Both the frequency and multiplicity of skeletal defects increased as the dose was raised. The stage dependency of defects in the orofacial bones was markedly apparent. Treatment on Day 10.5 resulted in shortened maxilla in all survivors, while treatment on either Day 11 or 11.5 resulted in a high frequency of reduction in the length of both upper and lowerjaws. Treatment on the 12th day resulted in a very low incidence of effect on the maxilla (4%) but a high frequency (88–100%) of reduction and deformation of the mandible. This enhanced susceptibility of individual facial bones at different stages of development with virtually no overlap provides an experimental model to study cellular phenomena underlying normal and abnormal facial development. Ribavirin, both in vivo and in vitro, inhibited embryonic DNA synthesis. The inhibition was transitory and did not seem to be directly related to the embryolethal activity of the drug. Although the role of metabolic inhibition in precipitating teratogenesis is not clear, cytotoxic action of ribavirin against proliferating limb bud mesenchymal cells is directly associated with the origin of limb deformities.     

Potassium prevention of lithium-induced loss of water and sodium in rats

Lithium was administered to rats with the food in amounts leading to serum lithium concentrations of 0.6–0.8 mmol/liter. The animals had free access to water and 0.46 m NaCl solution. Some of the rats were given a diet to which 500 mmol/kg of either potassium chloride or potassium phosphate had been added, while other rats received food without extra potassium. In the course of 3 weeks the water and sodium intakes increased in all groups, but they increased significantly less in the rats given potassium than in those given no extra potassium. During 6 hr of fluid deprivation the rats given extra potassium showed significantly less urine production, weight loss, and increase in serum sodium concentration. When access to NaCl solution was withdrawn the animals given extra potassium were almost unaffected whereas within 14 days the animals not given extra potassium developed lithium intoxication characterized by weight loss, decreased intake of water and food, lowered serum sodium concentration, and increased serum lithium concentration; two of the seven rats in this group died. In none of the experiments was there any difference between rats given potassium chloride and rats given potassium phosphate. The data indicate that a high potassium content in the food prevents lithium-induced renal inability to retain water and sodium.     

Tissue distribution and retention of cadmium in rats during postnatal development: Minimal role of hepatic metallothionein

Since liver concentration of metallothionein in 4-day-old rats is 20 times greater than that in 70-day-old adults, experiments were performed to determine whether the high concentration of metallothionein in the liver of newborn rats results in a greater distribution of cadmium to the liver and in subsequent lower concentration of cadmium in other tissues of the newborn in comparison to the adult, and if the retention of cadmium in the various tissues is affected. The concentration of cadmium in the liver of 4-day-old rats was only 30% higher than that in adult rats 2 hr after administration (1 mg/kg, iv). Even though there is no age difference in the renal concentration of metallothionein, the initial concentration of cadmium in the kidney of 4-day-old rats was half that in adult rats. However, cadmium was three to six times more concentrated, 2 hr after cadmium administration, in the spleen, bone, brain, testes, and muscle of newborn than in adult rats. Over a 19-day period, cadmium was similarly retained in the liver, kidney, and lung of newborn and adult rats, but the rate of cadmium elimination was higher from the heart and lower from the testes of newborn rats when compared to adults. These results indicate that metallothionein does not appear to play a major role in the tissue distribution and tissue retention of cadmium. The higher concentration of cadmium in some target organs, such as testes, kidneys, and lungs, of the newborn rat may have significant toxicological implications.