Melanie F. Struve

Neurotoxicity of manganese chloride in neonatal and adult CD rats following subchronic (21-day) high-dose oral exposure.

The purpose of this study was to evaluate the relative sensitivity of neonatal and adult CD rats to manganese‐induced neurotoxicity. Identical oral manganese chloride (MnCl2) doses (0, 25, or 50 mg kg−1 body wt. day−1) were given to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 through 21) and to adult male rats for 21 consecutive days. The MnCl2 doses administered to neonates were ca. 100‐fold higher than those resulting from the consumption of an equivalent volume of rats milk. Rats were assessed using similar behavioral and neurochemical evaluations. Several statistically significant changes occurred in Mn‐exposed rats relative to control animals. Neonates given the high dose of MnCl2 had reduced body weight gain. An increased pulse‐elicited acoustic startle response amplitude was observed in neonates from both MnCl2 treatment groups on PND 21. Increased striatal, hippocampal, hindbrain and cortical Mn concentrations were observed in all Mn‐exposed neonates on PND 21. Increased hypothalamic and cerebellar Mn concentrations were also observed on PND 21 in neonates from the high‐dose group only. Increased striatal, cerebellar and brain residue Mn concentrations were observed in adult rats from the high‐dose group. Increased striatal dopamine and 3,4‐dihydroxyphenylacetic acid levels were observed only in PND 21 neonates from the high‐dose group. No treatment‐related changes were observed in clinical signs, motor activity (assessed in neonates on PND 13, 17, 21 ± 1 and in adults), passive avoidance (assessed in neonates on PND 20 ± 1 and in adults) or neuropathology (assessed in PND 21 neonates only). The results of our experiment suggest that neonates may be at greater risk for Mn‐induced neurotoxicity when compared to adults receiving similar high oral levels of Mn. Copyright

Fertility and developmental neurotoxicity effects of inhaled hydrogen sulfide in Sprague–Dawley rats

In this study, we examined whether perinatal exposure by inhalation to hydrogen sulfide (H2S) had an adverse impact on pregnancy outcomes, offspring prenatal and postnatal development, or offspring behavior. Virgin male and female Sprague-Dawley rats (12 rats/sex/concentration) were exposed (0, 10, 30, or 80 ppm H2S; 6 h/day, 7 days/week) for 2 weeks prior to breeding. Exposures continued during a 2-week mating period (evidence of copulation = gestation day 0 = GD 0) and then from GD 0 through GD 19. Exposure of dams and their pups (eight rats/litter after culling) resumed between postnatal day (PND) 5 and 18. Adult male rats were exposed for 70 consecutive days. Offspring were evaluated using motor activity (PND 13, 17, 21, and 60+/-2), passive avoidance (PND 22+/-1 and 62+/-3), functional observation battery (PND 60+/-2), acoustic startle response (PND 21 and 62+/-3), and neuropathology (PND 23+/-2 and 61+/-2). There were no deaths and no adverse physical signs observed in F0 male or female rats during the study. A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation, and the average number of implants per pregnant female. Exposure to H2S did not affect pup growth, development, or performance on any of the behavioral tests. The results of our study suggest that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat at occupationally relevant exposure concentrations (< or =10 ppm).

Respiratory Tract Responses in Male Rats Following Subchronic Acrolein Inhalation

The goal of this study was to characterize the respiratory tract toxicity of acrolein, including nasal and pulmonary effects, in adult male F344 rats. Animals underwent whole-body exposure to 0, 0.02, 0.06, 0.2, 0.6, or 1.8 ppm acrolein for 6 hr/day, five days/week for up to 65 exposure days (13 exposure weeks). Respiratory tract histopathology was evaluated after 4, 14, 30, and 65 exposure days, as well as 60 days after the end of the 13 week exposure. Acrolein exposure was associated with reduced body weight gain. Rats exposed to ≥ 0.06 ppm acrolein had depressed terminal body weights when compared with air-exposed controls. Histologic evaluation of the nasal cavity showed olfactory epithelial inflammation and olfactory neuronal loss (ONL) following exposure to 1.8 ppm acrolein. Moderately severe ONL in the dorsal meatus and ethmoid turbinates occurred within four days while septal involvement developed with ongoing exposure. A rostral-caudal gradient in lesion severity was noted, with the anterior portion of the nasal cavity being more severely affected. Acrolein exposure was associated with inflammation, hyperplasia, and squamous metaplasia of the respiratory epithelium. The lateral wall was amongst the most sensitive locations for these responses and increased respiratory epithelial cell proliferation occurred at this site following 4 to 30 days of exposure to ≥ 0.6 ppm acrolein. The NOAEL for nasal pathology seen in this study was 0.2 ppm acrolein.

Brain Manganese Concentrations in Rats Following Manganese Tetroxide Inhalation are Unaffected by Dietary Manganese Intake

Manganese-deficient individuals have decreased manganese elimination. This observation has prompted suggestions that relative manganese deficiency may increase the risk for manganese neurotoxicity following inhalation exposure. The objective of this study was to determine whether dietary manganese intake influences the pharmacokinetics of inhaled manganese tetroxide (Mn3O4). Postnatal day (PND) 10 rats were placed on either a low (2 ppm), sufficient (10 ppm), or high-normal (100 ppm) manganese diet for 2 months. Beginning on PND 77 +/- 2, male littermates were exposed 6 h per day for 14 consecutive days to 0, 0.042, or 0.42 mg Mn3O4/m3. End-of-exposure tissue manganese concentrations and whole-body 54Mn elimination rates were determined. Tissue manganese concentrations were dependent on the dietary intake of manganese, thus confirming that altered hepatic manganese disposition or metabolism occurred. Male rats given 100 ppm manganese diet developed increased manganese concentrations in the femur, liver, and bile and had elevated whole-body 54Mn clearance rates when compared to animals given 2 ppm manganese diet. Male rats exposed to 0.42 mg Mn3O4/m3 had increased manganese concentrations in the olfactory bulb, lung, liver, and bile when compared to air-exposed male rats. A significant interaction between the concentration of inhaled Mn3O4 and dietary manganese level was observed only with the end-of-exposure liver manganese concentration. Our results indicate that animals maintained on either a manganese-deficient or high manganese diet do not appear to be at increased risk for elevated brain manganese concentrations following inhalation exposure to high levels of Mn3O4.

Neurotoxicological effects associated with short-term exposure of Sprague-Dawley rats to hydrogen sulfide.

Although hydrogen sulfide (H2S) is a known neurotoxic hazard, only a limited number of experimental animal studies have examined its neurochemical or behavioral effects. Our aim was to determine if short-term inhalation exposure of rats to H2S would result in altered brain catecholamnine levels or impaired learning and memory. Three groups of adult male CD rats were tested; two groups were exposed by nose-only inhalation (0, 30, 80, 200, or 400 ppm H2S) and one group was exposed by whole-body inhalation (0, 10, 30, or 80 ppm H2S) for 3 h per day forfive consecutive days. The first group (n = 10 rats per concentration) was tested immediately following each daily nose-only H2S exposure for spatial learning with a Morris water maze. Core body temperatures were also monitored in these animals during and after the last H2S exposure. The second group of rats (n = 10 rats per concentration) was tested for spontaneous motor activity immediately following the fifth exposure. These rats were then euthanized and striatal, hippocampal, and hindbrain catecholamnine levels determined. A third group of rats (n = 5-7 rats per concentration) was pretrained on a multiple fixed- interval (FI) schedule and exposed whole-body. Daily performance on the FI schedule was compared for the week pre-exposure, for the exposure week immediately following daily exposures, and for the week postexposure. We observed significant reductions in motor activity, water maze performance, and body temperature following exposure only to high concentrations (> or = 80 ppm) of H2S. Exposure to H2S did not affect regional brain catecholamine concentrations or performance on the FI schedule. Additional studies using other measures of behavior and longer-term exposure to H2S may be required to more definitively address conditions under which H2S exposure results in behavioral toxicity.

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats.

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offsprings anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring.

Neurotoxicological Evaluation of Ethyl Tertiary‐Butyl Ether Following Subchronic (90‐day) Inhalation in the Fischer 344 Rat

The purpose of this study was to evaluate whether repeated 6‐h exposure (65 exposures over a 14‐week period) of male and female Fischer‐344 rats (n = 12 rats/sex/concentration) to ethyl tertiary‐butyl ether (ETBE) atmospheres at 500, 1750, or 5000 ppm would result in neurotoxicity. Neurotoxicity was assessed by a blinded functional observational battery (FOB), motor activity, and terminal neuropathology. Motor activity was assessed 4 days prior to ETBE exposure and following 20, 42, and 65 days of exposure. The FOB was assessed 4 days prior to ETBE exposure and following 1, 6, 10, 20, 42, and 65 days of exposure. Transient ataxia, a sign of narcosis, was noted in male rats immediately following the 6‐h exposure to 5000 ppm ETBE. Statistically significant treatment effects on motor activity were not observed. Minor changes in grip strength and hindlimb splay were observed; however, none demonstrated a dose–response relationship or a consistent pattern of neurological dysfunction. No gross or microscopic abnormalities were observed in the central, peripheral, or autonomic nervous systems of rats exposed to 5000 ppm ETBE. No statistically significant differences in brain weight or size were observed in ETBE‐exposed rats. A statistically significant increase in body weight was observed in female rats exposed to 5000 ppm following 42 and 65 exposure days. Although ataxia was a common feature of acute ETBE neurotoxicity in rats following high‐level exposure, adverse neurological effects are not expected in the general public at the anticipated exposure levels associated with automotive refueling.

Design and evaluation of an olfactometer for the assessment of 3-methylindole-induced hyposmia.

Few studies objectively evaluate olfactory function in animals following exposure to chemicals that induce nasal toxicity. An olfactometer capable of generating a reproducible olfactory stimulus and measuring an odorant-cued behavioral response was developed for rats from a commercially available two-way shuttle box. The box was modified to deliver the test odorant, acetaldehyde, to either of two chambers separated by a physical barrier consisting of a downward-directed airwall sandwiched between two exhaust panels. Male Fisher 344 rats were trained with either a coupled odorant- or tone-cued active avoidance paradigm in order to compare auditory-cued versus olfactory-cued learning and memory. Odorant-cued animals had faster acquisition and longer retention of the avoidance behavior than tone-cued animals. Animals given the model olfactory toxicant 3-methylindole (3-MI, 400 mg/kg, ip) had reduced odorant-cued avoidance, while no effect on tone-cued behavior was observed. In a follow-up study, additional odorant-trained rats were dosed with 0, 100, 200, or 300 mg/kg of 3-MI ip and olfactory function reassessed 6 days later. Histopathologic evidence of moderate to severe olfactory epithelial damage was observed in all rats 7 days after 3-MI administration. Only the highest 3-MI dose (300 mg/kg) was associated with a significant reduction in odor-cued avoidance behavior as compared to that seen in control. These results indicate that use of this olfactometer can provide a functional assessment of chemically induced olfactory toxicity and complements more routine nasal pathology.

Development of a Mouse Whole-Body Exposure System from a Directed-Flow, Rat Nose-Only System

AbstractIn many inhalation exposure experiments, such as pharmacokinetic studies in unrestrained pregnant animals, it is desirable to expose unrestrained animals and to remove animals from the exposure system at intermediate time points. A two-tiered, 32-port, directed-flow, nose-only exposure system was modified with extended 0.635-mm stainless steel inlet tubes to create a whole-body, modified nose-only (WB-MNO) exposure system. Individual pregnant CD-1 mice held within a rat nose-only tube were exposed to a well-mixed methanol (MeOH) atmosphere. The volume of an individual mouse (∼30 ml) constituted approximately 5% of the 600-ml tube volume. Maternal MeOH pharmacokinetics were obtained on gestational day 8 following 6-h WB-MNO MeOH exposures at either 10,000 or 15,000 ppm. Results from these WB-MNO exposures were compared with a 6-h, 15,000 ppm MeOH exposure using a Hinners-type 1-m3 whole-body inhalation chamber (WB-H). The WB-MNO exposure atmosphere was produced using a wick generator heated to 29-3...