Thomas J. Sobotka

A developmental test battery for neurobehavioral toxicity in rats: a preliminary analysis using monosodium glutamate calcium carrageenan, and hydroxyurea.

Abstract A test battery for evaluating developmental neurobehavioral toxicity was designed to attempt to meet both the general requirements of a sound test system, namely, comprehensiveness, usability, and sensitivity and the specific requirements promulgated by Britain, France, and Japan as part of their reproductive guidelines for new drugs. Monosodium glutamate (MSG) and calcium carrageenan (CC) were used to obtain preliminary data with this test battery using continuous dietary exposure from prior to conception through 90 days of postnatal life. Observations were made on reproduction and on the physical and behavioral development of the offspring. Three dose levels were used with each of the food additives and the data from these subjects were compared to data from normally fed (negative) controls and a positive control group exposed to 550 mg/kg of hydroxyurea on the 12th day of gestation. Differences between MSG and negative control groups were observed in swimming development, open field, active and passive avoidance testing. Effects observed in the CC groups were inconsistent and not dose related. Prenatal treatment with hydroxyurea produced delayed startle and swimming development and reduced open field rearing activity. This pattern of effects was less than expected indicating that hydroxyurea is an adequate, but not optimal, positive control manipulation. It appears that the test protocol used in this research could, with some modification, serve as a usable screening technique for developmental neurobehavioral toxicity.

Neonatal malnutrition:: neurochemical, hormonal and behavioral manifestations

Abstract An effective state of malnutrition was imposed on rat pups throughout their postnatal period of development by feeding the lactating dams a 12% casein diet. Controls were fed a 24% casein diet. The malnourished pups exhibited such signs of retarded physical development as reduced body growth and delayed eye opening. At weaning brain weights were reduced and regional brain analysis revealed distortions of chemical composition. These changes included a general reduction of cholesterol concentration, decreased cerebellar DNA content (indicative of cell number), and reduced telencephalic and brain stem protein: DNA ratios (reflecting cell size). The cerebellum also displayed reduced AChE activity suggesting an appreciable effect on its synaptic elements, particularly involving the cholinergic system. Brain stem concentrations of 5-HT and 5-HIAA were increased, implying activation of the central serotonergic neurohumoral system. Possibly related to this, a concomitant increase of adrenal corticosterone was revealed, implicating the additional involvement of the pituitary-adrenal axis in the consequences of postnatal malnutrition. Behaviorally, the previously malnourished weanlings displayed a state of heightened ‘emotionality’. Their performance in a 2-way shuttle task was also markedly abnormal. It is proposed that the enhanced activation of the brain stem serotonergic system may, at least partially, underlie the state of heightened ‘emotionality’, which is the most characteristic behavioral manifestation of perinatal malnutrition.

Psychophysiologic effects of early lead exposure.

In several separate experiments neonatal rats were intubated daily with 9, 27 or 81 mg lead acetate/kg of body weight throughout their 3-week postnatal period of development. Based on average body weights, the total daily lead intake was 0.156, 0.454 or 1.384 mg lead per animal, respectively (in addition to normal lead intake from the environment). Subtle and specific behavioral changes, involving an inability to attenuate inappropriate behavior in a two-way shuttle or a habit-reversal operant task, occurred in offspring following exposure to a minimum of 0.454 mg lead per day. The specificity of this central dysfunction was such that motor activity was normal, stress responsiveness remained unaffected and simple learning ability was comparable to that of controls. The only indication of a central neurochemical modification accompanying this behavioral defect was a tendency for telencephalic acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities to be depressed, suggesting a possible involvement of the cholinergic system. Steady-state levels of brain monoamines were unaltered. The experimental weanlings displayed an inhibition of blood delta-amino levulinic acid dehydratase (ALAD) activity, a parallel reduction in regional brain ALAD activity, a moderate reduction in hematocrit and hemoglobin and an increase in kidney weight. This latter effect occurred even at the lowest level of lead intake, 0.156 mg lead per day.

Biochemical and cytogenetic effects in rats caused by short‐term ingestion of aroclor 1254 or firemaster BP6

Male rats were fed either 0, 5, 50, or 500 ppm Aroclor 1254 (ARO) or Firemaster BP6 (FM) mixed in ground rat chow for 2, 3, or 5 wk. The relative biological response to the two chemicals sometimes varied with dose level. For example, for some parameters FM caused a greater effect than ARO at the 5 and 50 ppm levels, but not at the 500 ppm level, where, presumably, the maximum response was attained. The experimental design did not permit a totally unambiguous comparison of all the responses to ARO and FM. Some apparent differences in biochemical responses determined at sacrifice may have been partly due to the fact that, for the 3 wk study, rats were exposed to FM 48 hr longer than to ARO, and thus the comparison of ARO and FM toxiclties should be considered tentative. The following evaluations were made, based on the assumption that this 2 day difference had no effect. Relative kidney and testis weights, hematology, SGPT, SGOT, BUN, plasma corticosterone, rate of liver protein synthesis per gram of tissue...

Extracellular concentrations of amino acid transmitters in ventral hippocampus during and after the development of kindling

This study examined the possible involvement of amino acid release from ventral hippocampus in the establishment and maintenance of kindling in rats. Release of amino acids from ventral hippocampus was measured by microdialysis coupled with high-performance liquid chromatography. Samples were obtained by microdialysis perfusion of freely moving animals receiving deep prepiriform cortex (DPC) electrical stimulation. Samples of perfusate were collected before, during and after kindling was established. DPC kindling stimulation significantly increased concentrations of glutamate (Glu) and glycine (Gly) in perfusate from ventral hippocampus during kindling. Increased basal release of Glu was evident up to 30 days after the last electrical stimulation. We conclude that release of Glu and Gly in the ventral hippocampus may play an important role during establishment, but not in maintenance of kindling.

Developmental neurobehavioural toxicity of butylated hydroxytoluene in rats.

Abstract Butylated hydroxytoluene (BHT) was fed to rats throughout development (from before conception through to day 90 of postnatal life) at levels of 0, 0·125, 0·25 or 0·5% (w/w) in the diet. A similarly treated positive control group was injected on day 12 of gestation with 550 mg/kg of the antimitotic/embryotoxic drug hydroxyurea for reference. Offspring from all groups were reared by their natural dams and were evaluated in a battery of behavioural tests from day 3 to day 90 after birth. BHT at 0·5% in the diet reduced the body weights of dams and of offspring during early development and increased offspring mortality (to 39%) up to 30 days of age. This dose delayed eyelid opening, surface-righting development and limb co-ordination in swimming in males, and reduced female open-field ambulation; however, no significant effects were found after weaning. The lower doses of BHT produced some irregularities in maternal weight (0·25% an increase and 0·125% a decrease) but had no effect on the body weights of offspring. BHT at 0·25% of the diet increased pre- and periweaning mortality (23%), but neither this dose nor the 0·125% dose had any effect on physical or behavioural development or on post-weaning behavioural performance. The positive control group treated with hydroxyurea showed reduced growth prior to weaning, reduced adult brain weight and a slight but nonsignificant increase in pre- and periweaning mortality (10%). This group also exhibited delayed eyelid opening, delayed forward locomotor development and limb co-ordination during swimming, but showed no effects on postweaning behavioural performance. The BHT findings are consistent with the existing toxicological literature that BHT is toxic to growing rodents at doses of 0·25 or 0·5% of the diet with marginal effects at 0·125% of the diet. The behavioural data expand the picture of BHTs toxicity, but do not suggest any disproportionate or special toxicity of BHT for the central nervous system.

Developmental toxicity and psychotoxicity of FD and C red dye No. 40 (allura red AC) in rats.

Adult Sprague-Dawley rats were fed diets containing FD and C red dye No. 40 for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups were: FD and C red dye No. 40 as 0.0, 2.5, 5.0 or 10.0% of the diet, and a positive control group treated with the toxin hydroxyurea on days 2-10 of life with 50 mg/kg/day given s.c. as a positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery. Additional measures were weight, food consumption, physical landmarks of development, and brain weight. Red-40 significantly reduced reproductive success, parental and offspring weight, brain weight, survival, and female vaginal patency development. Behaviorally, R40 produced substantially decreased running wheel activity, and slightly increased postweaning open-field rearing activity. Overall, R40 produced evidence of both physical and behavioral toxicity in developing rats at doses of up to 10% of the diet.

High doses of aspartame have no effects on sensorimotor function or learning and memory in rats

Acute or repeated (14 days) intragastric administration of L-d-aspartyl-L-phenylalanine methyl ester (aspartame) suspended in saline and Tween-80 in doses of up to 1,000 mg/kg had no significant effect in male Fischer-344 rats on routine measures of sensorimotor function, including spontaneous motor activity, acoustic startle reflex and prepulse inhibition. Other experiments found that aspartame (500 or 1,000 mg/kg) had no significant effect on acquisition of passive or active avoidance or a spatial, reference memory task in the Morris water maze. A series of separate studies found that aspartame had no effects in rats fasted 24 hours prior to testing, or if it was suspended in carboxymethylcellulose or administered by the intraperitoneal route. Under the conditions of these experiments, large doses of aspartame have no significant neurobiological effects in adult rats as measured by procedures known to be sensitive to the neurobiological effects of neurotoxicants, including convulsants, organochlorine insecticides and heavy metals.

Tartrazine and the developing nervous system of rats

Rat dams were exposed to the artificial food color tartrazine (FD&C Yellow no. 5) at dietary levels of 0, 1, and 2% during gestation and lactation. The experimental offspring were continued on the same diets for approximately 3 months after weaning. No adverse physical or behavioral effects were noted in the dams. Fetal development and postnatal viability of the offspring were also normal. The only effect on postnatal development of the central nervous system (CNS) was a small transient change in neuromotor clinging ability of female offspring. The limited effect of tartrazine on the CNS was further evidenced by the facts that (1) the neurobehavioral profiles of the experimental weanlings revealed no significant abnormalities, and (2) morphochemical analysis of brain tissue, as well as brain weights, revealed no abnormalities. Tartrazine did appear to exert more general signs of toxicity in the offspring--namely, depressed body weight, an apparent reduction in thymus weight, and a slight elevation of red blood cells and hemoglobin.

A developmental toxicity and psychotoxicity evaluation of FD and C Red Dye #3 (erythrosine) in rats

Two experiments were conducted to evaluate FD and C Red Dye #3 for its developmental toxicity and psychotoxicity. Adult Sprague-Dawley rats were fed diets containing the dye for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups for Experiment 1 were Red Dye #3 as 0.0, 0.25, 0.5, or 1.0% of the diet (w/w), and a positive control group treated with the toxin hydroxyurea on days 2–10 of life (50 mg/kg/day, s.c.); Experiment 2 was a replication of Experiment 1 with the same dose groups, but without the positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery, plus weight, food consumption, physical landmarks of development, and brain weight. Red-3 produced no reductions in parental or offspring weight or food consumption. Red-3 significantly increased preweaning offspring mortality in the first experiment, but not in the second. Behaviorally, Red-3 produced no dose-dependent effects that replicated across the two experiments. It was concluded that no evidence was obtained that dietary exposure to FD and C Red Dye #3 (erythrosine) is psycho toxic to developing rats.