Repeated analysis of semen parameters in beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin.

Sperm analyses are often incorporated into reproductive toxicity studies in rats. Due to the relative ease of collecting multiple samples throughout a study, semen analysis in non-rodents such as dogs offers the opportunity to assess potential development of functional effects of compounds on male reproduction over time. In the present study, semen parameters were evaluated in beagle dogs during and at termination of a chronic toxicity study with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male dogs received 0, 10, 40, or 120 mg/kg orally in gelatin capsules for up to 104 weeks (n = 10/group). After 52 weeks of dosing, 3 dogs/group were euthanized, and 2/group were withdrawn from treatment for a 12-week reversal period and euthanized at Week 64. The remaining 5/group continued treatment until Week 104. Semen was collected from all animals for 3 consecutive weeks prior to termination of the 52-week animals (Weeks 50, 51, 52) for analysis of sperm parameters, using manual methods of evaluation. Semen was collected from the remaining animals at Weeks 64, 78, 91, and 104, and was analyzed. At necropsy, testes, epididymides, and prostates were weighed and evaluated histologically, and epididymal sperm counts were determined. Serum cholesterol was decreased 25--60% at all doses during the study. There were no drug-related differences in semen volume and color, total sperm count, and sperm concentration, morphology, progressiveness, and percent motility during treatment with atorvastatin. There were also no effects on reproductive organ weights or histopathology, and no effects on epididymal sperm count. Thus, incorporation of semen analyses into this study allowed the evaluation of potential male reproductive effects in dogs at multiple time points during the study. Statistical power calculations demonstrated acceptable statistical power (> 80%) for semen sperm count, concentration, morphology, and motility with group sizes of 8--10 animals, and for semen sperm count and concentration or epididymal sperm count with group sizes of 3--5 animals, using the methodology described in this paper.

Fertility and general reproduction studies in rats with the HMG-CoA reductase inhibitor, atorvastatin.

Fertility and reproduction studies were conducted in rats with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male rats received vehicle (0.5% methylcellulose) or atorvastatin at 20, 100, or 175 mg/kg by oral gavage for 11 weeks prior to mating with untreated females; treatment continued throughout mating and until necropsy on Day 115. An untreated control group of males was also included in the same procedures. Dose-related body weight gain suppressions of 17 and 25%, and food consumption suppressions of 7 and 16%, occurred during the 11-week premating treatment period at 100 and 175 mg/kg, respectively, compared with vehicle controls. There were no treatment-related effects on testes, epididymides, or accessory organs weights, testicular or epididymal sperm counts, sperm motility, or sperm morphology during Week 15 of treatment. Plasma drug concentrations during Week 15 increased with dose to a Cmax of 1820 +/- 1020 ng eq/ml at 175 mg/kg. There were no effects on copulation or fertility indices, number of days to mating, or female reproductive parameters (number of implants, live fetuses, or pre- and postimplantation loss). In the female fertility study, female rats received vehicle (0.5% methylcellulose) or atorvastatin at 20, 100, or 225 mg/kg by oral gavage for 2 weeks prior to mating with untreated males; treatment continued throughout mating and until Gestation Day 7. Sperm-positive females were sacrificed on presumed Gestation Day 13 to 15 for evaluation of reproductive parameters. Body weight gain in atorvastatin groups was comparable to controls during the premating period, but was suppressed by 35% at 225 mg/kg during the treatment period of gestation (Days 0-8), and was significantly increased at 225 mg/ kg during the posttreatment period of gestation (Days 8-13). Plasma drug concentrations on premating treatment Day 14 increased with dose to a Cmax of 7030 +/- 3680 ng eq/ml at 225 mg/ kg. The mean number of estrous cycles, copulation and fertility indices, number of days to mating, and number of viable litters were comparable between groups. In addition, term sacrifice parameters (number of corpora lutea, implants, live fetuses, pre- and postimplantation loss) were not significantly different between groups. Thus, these studies demonstrate no adverse effects of atorvastatin on fertility and reproduction in rats at doses up to 175 and 225 mg/kg in males and females, respectively, and 20 mg/kg was a no-effect dose.

Sperm motion parameters in vas deferens and cauda epididymal rat sperm.

Motion parameters were compared in rat sperm isolated from the distal vas deferens and the cauda epididymidis. Motion parameters were also compared in 20 microns and 50 microns deep muCellTM chambers using vas deferens sperm. Video recorded samples were analyzed manually for motility, and analyzed by a computer automated sperm analysis (CASA) system for motility, curvilinear velocity, linearity, mean and maximum amplitude of lateral head displacement (ALH), and beat/cross frequency using two versions of CellSoftTM (Series 3,000 and Series 4,000). Motility, linearity, and beat/cross frequency were not significantly different between sperm from vas deferens and cauda epididymidis, while velocity and ALH values were slightly greater in sperm from vas deferens than from cauda epididymidis. Sperm motility and linearity were not significantly different when analyzed in 20 microns and 50 microns mu CellTM chambers. Velocity and ALH values were slightly greater in 20 microns than in 50 microns chambers, and beat/cross frequency was slightly lower in 20 microns than in 50 microns chambers. Sperm motility was significantly greater when determined manually than when determined with the Series 3,000 but manually determined sperm motility was only slightly greater than motility determined with the Series 4,000. Several sperm motion parameters differed significantly between the Series 3,000 and Series 4,000 (curvilinear velocity, mean and maximum ALH, linearity, and beat/cross frequency) but the relative variability of the systems was comparable. Compared with manual determinations, the Series 3,000 overestimated and the Series 4,000 underestimated the number of cells analyzed for motility. Therefore, differences existed between manual and CellSoft (Series 3,000 and 4,000) analysis of sperm motility and number of cells, and between CellSoft systems in the analysis of sperm motion parameters. However, only minimal differences in sperm motion parameters were observed between the vas deferens and cauda epididymidis, and between 20 microns and 50 microns deep muCell chambers.

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. ILSI Risk Science Institute Expert Working Group on Sperm Evaluation.

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Developmental toxicity study in rats treated with the anticonvulsant, ralitoline.

The developmental toxicity of the anticonvulsant compound, ralitoline, was investigated in Sprague-Dawley rats administered oral doses of 0, 15, 60, 120, 180, or 240 mg/kg on days 6 through 15 of gestation. An untreated control group and a vehicle control group pair-fed to the high dose group were included. Maternal and fetal parameters were evaluated on day 21 of gestation. Fetuses were examined for external, visceral, and skeletal malformations and variations. Maternal death occurred at 180 and 240 mg/kg. Dose-dependent decreases in body weight, food consumption, and water consumption were observed at 60 mg/kg and above. Body weight gain during treatment was similar in the pair-fed and 240 mg/kg groups. Dose-related CNS signs (hypoactivity, ataxia, prostration, and/or convulsions) were observed at 60 mg/kg and above. Decreased numbers of live fetuses and increased postimplantation loss were observed in a dose-related manner at 120, 180, and 240 mg/kg while no changes occurred in pair-fed controls. Fetal body weights and placental weights were decreased in pair-fed controls and in the 120, 180, and 240 mg/kg groups. The percent fetuses per litter, and the percent litters with external/visceral malformations, were significantly increased at 120, 180, and 240 mg/kg compared with vehicle and pair-fed controls. Dose-related increases in cardiovascular malformations, specifically of the aortic arch (interrupted, stenotic, extra vessel), were apparent at 120 mg/kg and above. The incidence of skeletal variations was increased at 120 mg/kg and above.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity of the HMG-CoA reductase inhibitor, atorvastatin, in rats and rabbits.

The developmental toxicity of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin, was investigated in pregnant rats and rabbits given daily oral doses during organogenesis. Rats received 0, 10, 100, or 300 mg/kg on days 6-15 of gestation, and rabbits received 0, 10, 50, or 100 mg/kg on days 6-18 of gestation. Maternal and fetal parameters were evaluated on day 20 (rats) or 29 (rabbits) of gestation. Live fetuses were examined for external, visceral, and skeletal malformations and variations. At 300 mg/kg in rats, 1 treatment-related death occurred on day 12 of gestation, and maternal body weight gain and food consumption were decreased during treatment (43% and 23%, respectively). In addition, 1 animal at 300 mg/kg had total litter resorption. Increased postimplantation loss (not statistically significant) and slightly decreased fetal body weight (statistically significant only in males) were also observed at 300 mg/kg. There were no significant differences between treated and control groups in the incidence of fetal malformations or variations. No maternal or developmental toxicity was observed in rats at 10 or 100 mg/kg. In rabbits, marked maternal toxicity (7 deaths, body weight loss during and after treatment, and decreased food consumption) and abortion occurred at 100 mg/kg. At 50 mg/kg, maternal toxicity (2 deaths and 72% body weight gain suppression) and abortion also occurred. There were no treatment-related effects on live litter size or sex ratio. At 50 and 100 mg/kg, nonstatistically significant increases in postimplantation loss and decreases in gravid uterine weight were observed, and at 100 mg/kg, decreases in fetal body weight were observed relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Female reproductive and developmental toxicology: overview and current approaches.

In recent years, concern about possible female reproductive and developmental toxicity due to environmental contaminants, such as PCBs, has been growing. Because this area of toxicology had not been emphasized prior to this time, there are many gaps in current knowledge about female developmental and reproductive toxicology and only a limited number of validated tests to assay effects of toxicants on various parts of the reproductive and developmental cycle. This article reviews the current state of knowledge on this topic and also explores a variety of techniques for assessing female reproductive and developmental toxicity. These include an assay of the state of intercellular communication among the embryo, fetus and placenta; protocols for assessing toxicity in early pregnancy; and techniques for evaluating the role of glutathione in protecting the conceptus from xenobiotics.

Fertility and perinatal/postnatal studies in rats with the angiotensin-converting enzyme inhibitor, quinapril.

Quinapril, an inhibitor of angiotensin-converting enzyme (ACE) and an antihypertensive agent, was evaluated in rats for effects on fertility, reproduction, and perinatal and postnatal development. In a fertility study, male rats were treated by gavage for 60 days prior to and during mating and female rats were treated by gavage for 14 days prior to mating, during mating and gestation, and during lactation with doses of 0, 10, 50, or 100 mg quinapril/kg body wt. There were no significant effects on body weight, food consumption, fertility indices, fetal development, or neonatal growth, survival, development, behavior, or reproduction. In a perinatal/postnatal study, administration of quinapril to females at doses of 25, 75, or 150 mg/kg during late gestation and lactation had no effects on parturition, lactation, or postnatal development, but a significant decrease in neonatal body weight during the suckling period was observed at all doses. In a subsequent study, female rats were given 150 mg/kg during late gestation, lactation, or late gestation and lactation. No adverse effects were seen in the dams or the offspring, and no reduction in neonatal body weight was observed. Kidneys from pups whose mothers received quinapril during gestation and/or lactation had minimal juxtaglomerular cell hypertrophy, characteristic of treatment with ACE inhibitors. Low levels of quinaprilat (the major and pharmacologically active metabolite of quinapril) were detected in fetal blood and in neonatal blood, indicating offspring exposure to quinapril. Milk quinaprilat concentrations were 3-5% of the plasma concentrations 3-5 hr after dosing. These studies demonstrate no adverse effects of quinapril on fertility, reproduction, or perinatal and postnatal development.

Developmental toxicity of pentostatin (2'-deoxycoformycin) in rats and rabbits.

The developmental toxicity of the potent adenosine deaminase (ADA) inhibitor, pentostatin (2'-deoxycoformycin), was investigated in pregnant rats and rabbits administered daily iv doses during organogenesis. Rats received 0, 0.01, 0.10, or 0.75 mg/kg on gestation days 6-15 and rabbits received 0, 0.005, 0.01, or 0.02 mg/kg on gestation days 6-18 and maternal and fetal parameters were evaluated on gestation day 21 (rats) or 30 (rabbits). Live fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal body weight gain and food consumption were significantly suppressed at doses of 0.10 and 0.75 mg/kg during the treatment period but returned to control levels during posttreatment. Increased postimplantation loss and decreased numbers of live fetuses, litter size, and fetal body weight were observed at 0.75 mg/kg. A statistically significant increase in the incidence of vertebral malformations occurred at 0.75 mg/kg. The incidence of certain skeletal variations (extra presacral vertebrae, extra ribs, hypoplastic vertebrae) was also increased at 0.75 mg/kg. Ossification of cervical centra was reduced at 0.75 mg/kg compared with controls. In rabbits, marked maternal toxicity (death, body weight loss, and decreased food consumption) and reproductive toxicity (abortion and premature delivery) occurred in all pentostatin-treated groups. However, there were no significant effects on number of live fetuses, pre- or postimplantation loss, litter size, or fetal body weights in the animals with live litters. There was also no apparent increase in the incidence of malformations or variations in the live fetuses of pentostatin-treated rabbits. Thus, these studies demonstrate developmental toxicity of pentostatin in rats and rabbits, and teratogenicity in rats, at maternally toxic doses.

Excretion of high concentrations of cimetidine and ranitidine into rat milk and their effects on milk composition and mammary gland nucleic acid content.

The excretion of cimetidine and ranitidine into rat milk following single or multiple oral doses and the subsequent effects on their suckling pups and on milk composition and milk synthesis were investigated. Following a single dose of [3H]cimetidine, peak milk cimetidine concentrations were maintained from 1 until 4 hr, while plasma concentrations peaked at 10% of the milk level at 30 min and then declined. Multiple doses of cimetidine (18 or 180 mg/kg/day) on Days 13-16 of lactation led to milk cimetidine concentrations of 17 and 113 micrograms/ml. The milk/plasma ratios far exceeded the theoretical milk/plasma ratio of 2.0. Ranitidine concentrations in rat milk following ranitidine treatment (4.5 or 45 mg/kg/day) were also greater (6.8-15 times) than in plasma, but only slightly greater than the predicted ratio of 5.0. There were no changes in liver weight or in hepatic aminopyrine N-demethylase activity in the cimetidine- or ranitidine-treated dams or their pups. Cimetidine treatment had no effect on milk lipid, solid, or protein content, but at 180 mg/kg/day, caused a significant increase in milk lactose. The RNA/DNA ratio in the mammary gland was significantly increased by cimetidine, suggesting increased milk synthesis. Ranitidine had no effect on milk composition or on mammary gland RNA, DNA, or RNA/DNA. Therefore, high concentrations of cimetidine and ranitidine were excreted into rat milk, but no deleterious effects on the suckling pups, or the composition of the milk, or on the milk synthetic activity were observed.

Effects of nickel chloride on lactating rats and their suckling pups, and the transfer of nickel through rat milk.

The excretion of nickel into rat milk following subcutaneous (sc) doses of nickel chloride (NiCl2) and the effects on the lactating rat and her suckling pups were determined. Plasma and milk Ni concentrations increased in a dose-dependent manner 4 hr after single doses of 0, 10, 50, or 100 mumol NiCl2/kg to lactating rats, giving milk/plasma Ni ratios of 0.02. Peak plasma Ni concentrations were reached 4 hr after injection, while milk Ni increased until 12 hr and remained elevated at 24 hr. Dosing for 4 days at 50 or 100 mumol NiCl2/kg/day led to higher milk/plasma Ni ratios of 0.10. These doses of NiCl2 had no effect on body weight but caused decreased food consumption, thymic atrophy, and a small increase in hepatic lipid peroxidation in the dams. Significant alterations in milk composition, which were not due to decreased food consumption as determined in pair-fed rats, included increased milk solids (42%) and lipid (110%), and decreased milk protein (29%) and lactose (61%). NiCl2 treatment also caused significant decreases in mammary RNA content and the RNA/DNA ratio compared to both ad libitum-fed and pair-fed rats, indicating that milk synthetic activity was reduced by NiCl2. Pups suckling the NiCl2-treated dams had plasma Ni concentrations of 24 and 48 micrograms/liter in the 50 and 100 mumol/kg dose groups, respectively, and had decreased liver weight but no changes in hepatic lipid peroxidation or thymus weight. The results indicate that high doses of NiCl2 led to the excretion of Ni into rat milk and changes in milk quality and production. Reductions in liver weight in the suckling pups were also observed which may have been due to nickel exposure or to changes in milk composition.

Determination of diphenhydramine in rat milk and plasma and its effects on milk composition and mammary gland nucleic acids.

To study the excretion of diphenhydramine into rat milk, milk and plasma concentrations of diphenhydramine were determined in lactating rats after single or multiple oral doses. Four hours after a single dose of 40 or 100 mg/kg of diphenhydramine, milk concentrations of the drug averaged 0.30 and 2.2 micrograms/mL, respectively, in two experiments, and the milk:plasma ratios ranged from 4.4 to 7.5. Multiple doses did not significantly affect the plasma or milk concentrations or the milk:plasma ratios, which were similar to the theoretical milk:plasma ratio based on pH partitioning for this compound (i.e., 4.0). Although the concentration of diphenhydramine was higher in milk than in plasma, the estimated dose received by the pups (0.057 mg/kg/d) based on the milk concentrations was much lower than that given to the mother. Oral diphenhydramine treatment at doses which significantly reduced maternal food consumption had no effect on milk solid, lipid, protein, or lactose concentrations, nor on mammary gland RNA or DNA content, indicating that diphenhydramine did not adversely affect lactation.

Testicular toxicity and reduced Sertoli cell numbers in neonatal rats by di(2-ethylhexyl)phthalate and the recovery of fertility as adults.

Neonatal and adult rats (1, 2, 3, 6, and 12 weeks of age) were given five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (0, 10, 100, 1000, 2000 mg/kg) and histological changes in the testes were examined 24 hr after the last dose. Relative testis weights were reduced at doses of 1000 mg/kg in 1, 2, 3, and 6-week-old but not in 12-week-old rats, while doses of 2000 mg/kg were fatal to suckling rats and caused decreased relative testis weight but not death in 6- and 12-week-old rats. In neonatal rats (1 week old), DEHP (1000 mg/kg) caused a 35% decrease in Sertoli cell numbers while 2- and 3-week-old rats showed losses of spermatocytes but not of Sertoli cells. The 6- and 12-week-old rats showed loss of both spermatids and spermatocytes at 1000 and/or 2000 mg/kg. Total testicular zinc concentrations were decreased in 12-week-old but not in suckling (3-week) or weaned (6-week) rats. The results support the hypothesis that the Sertoli cell is the primary testicular target of phthalate ester toxicity since effects were observed at an age when only Sertoli cells were present. Fertility was assessed in mating trials in adult male rats after neonatal exposure to DEHP on Days 6-10. Although Sertoli cell number was reduced 24 hr after the last dose, the numbers were normal at 6 and 13 weeks of age. However, at 6 weeks there was a dose-related decrease in maturation of the spermatids in the tubules. There were no consistent changes in fertility, implantation rate, or numbers of live fetuses in untreated females mated with the DEHP-treated males. However, there were decreases in testis weight and testicular spermatid numbers at 13 and 19 weeks but not at 11, 12, 16, or 23 weeks of age. Therefore, a loss of Sertoli cells due to DEHP exposure neonatally did not affect the fertility of the rats as adults, but may have caused subtle effects on sperm production.

Stereoselectivity and regioselectivity of purified human glutathione transferases pi, alpha-epsilon, and mu with alkene and polycyclic arene oxide substrates.

The stereoselectivities of three biochemically distinct human glutathione transferases, the acidic isoenzyme (pi) purified from placenta and the basic (alpha-epsilon) and the near-neutral (mu) isoenzymes purified from liver, were determined with (+/-)-benzo(a)pyrene-4,5-oxide, pyrene-4,5-oxide, and (+/-)-styrene-7,8-oxide as substrates. Transferase mu was highly selective (greater than 95%) for reaction of glutathione with R-configured oxirane carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide, whereas transferase pi was highly stereoselective (greater than 95%) for S-configured epoxide carbon atoms of (+/-)-benzo(a)pyrene-4,5-oxide and pyrene-4,5-oxide. The basic transferases (alpha-epsilon) showed relatively low stereoselectivity with these polycyclic arene oxide substrates; glutathione reaction at R-configured oxirane carbons was preferred, but only by about 2-fold. With (+/-)-benzo(a)pyrene-4,5-oxide as substrate, transferases mu and alpha-epsilon were enantioselective for (4R,5S)-benzo(a)pyrene-4,5-oxide (about 6-fold), whereas transferase pi showed little enantioselectivity. With (+/-)-styrene-7,8-oxide as substrate, transferases mu and pi were selective for (7S)-styrene-7,8-oxide, but this enantioselectivity was not great (1.3- to 1.8-fold); enantioselectivity could not be accurately determined with alpha-epsilon due to the low enzymatic turnover. Transferase pi selectively catalyzed the reaction of glutathione with the benzylic oxirane carbon (C-7) of (+/-)-styrene-7,8-oxide whereas alpha-epsilon preferentially catalyzed reaction with the terminal epoxide carbon (C-8) atom.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland.

Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 +/- 23 micrograms/ml DEHP and 25 +/- 6 micrograms/ml MEHP (mean +/- SE), while the plasma contained less than 0.5 micrograms/ml DEHP and 75 +/- 12 micrograms/ml MEHP. The high milk/plasma ratio for DEHP (greater than 200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups.

Stereoselectivity of cytosolic glutathione S-transferases with arene and alkene oxide substrates in various tissues and isolated hepatic and pulmonary cells of the rabbit.

The specific activity and stereoselectivity of cytosolic glutathione S-transferases (GST) from various rabbit tissues and isolated cells were determined as an initial step in characterizing GST isoenzymes in the rabbit. Of the five tissues examined, liver cytosol had the highest specific GST activity with the polycyclic arene oxides (+/-)-benzo[a]pyrene 4,5-oxide (BPO), pyrene 4,5-oxide (PO) and (+/-)-benz[a]-anthracene 5,6-oxide (BAO), and kidney cytosol had the second highest. Lung, intestine and testis had relatively low activities with all three substrates. With the alkene oxide (+/-)-styrene 7,8-oxide (SO), testicular cytosol had the highest GST activity while liver cytosol had only half the activity of the testis. Cytosolic GST from liver and kidney were highly stereoselective for reaction of glutathione with the S-configured oxirane carbon atoms of BPO, PO and BAO, and all tissues but the intestine were enantioselective for (4R,5S)-BPO and (5S,6R)-BAO. With SO, the liver, kidney and testis preferentially catalyzed the reaction of glutathione with the benzylic carbon atom of (7S)-SO. There was virtually no enantioselectivity in lung cytosol with SO but a preference for reaction with (7R)-SO was noted in the intestine. The stereoselectivities found in the intestine with each of the four substrates were markedly different from the other tissues. Cytosol from isolated hepatocytes showed almost identical patterns of stereoselectivity with BPO and PO to those of whole liver cytosol. Similarly, the stereoselectivity of cytosol prepared from alveolar type II cells isolated from rabbit lung was the same as that of whole lung cytosol with these substrates, whereas cytosol of alveolar macrophages differed substantially from lung cytosol in both cases. There were marked differences in stereoselectivity of cytosol from freshly isolated Clara cells with BPO versus PO as substrate. With BPO, Clara cells were very similar to whole lung cytosol, but with PO they were not. The data are consistent with the differential tissue and cellular distribution of multiple GST isoenzymes in the rabbit.

Hepatic peroxisome proliferation and hypolipidemic effects of di(2-ethylhexyl)phthalate in neonatal and adult rats.

To determine the relative sensitivity of suckling rats as compared to adults to the effects of di(2-ethylhexyl) phthalate (DEHP), five daily oral doses of 0, 10, 100, 1000, or 2000 mg DEHP/kg body weight were given to male Sprague-Dawley rats beginning at 6, 14, 16, 21, 42, and 86 days of age. Twenty-four hours after the last dose, rats were sacrificed and plasma cholesterol and triglyceride levels and the activities of the hepatic peroxisomal enzymes, palmitoyl CoA oxidase and carnitine acetyltransferase, were determined. Suckling rats (1-3 weeks of age) suffered severe growth retardation at doses of 1000 mg/kg and death at 2000 mg/kg while older rats only showed decreased weight gain at 2000 mg/kg. Of particular interest was the lethality at doses of 1000 mg/kg at 14 days of age but not at 16 days or at other ages. Increases in relative liver weight and hepatic peroxisomal enzyme activities were similar in all age groups except the 14-day old group in which the increases were greater. Relative kidney weight was increased in 21-, 42-, and 86-day-old rats at the highest doses but not in younger rats. Hypolipidemia was observed only in 21-, 42-, and 86-day-old rats at doses of 1000 and 2000 mg/kg, while elevated plasma cholesterol levels were observed in 6- and 14-day-old rats at the 1000 mg/kg dose, possibly due to the dietary differences between suckling and weaned rats. The results suggest that neonatal and suckling rats are more sensitive to the lethal and growth retardation effects of DEHP than are adult rats, but the hepatic peroxisome proliferation is similar at all ages with the exception of a greater increase at 14 days of age.

Alteration of the redox state of NADPH and glutathione in perfused rabbit lung by paraquat.

The glutathione and NADP+ status of perfused rabbit lung was determined both before and after perfusion with 1 mM paraquat. The pulmonary glutathione redox state was similar to that of liver, having a glutathione/glutathione disulfide ratio of about 240. This ratio was lower in lungs perfused with glucose-free medium, a condition in which NADPH probably becomes limiting. Perfusion with paraquat significantly increased the pulmonary glutathione disulfide content, particularly in the absence of glucose, resulting in a glutathione/glutathione disulfide ratio of less than 100. NADP+ levels in rabbit lung were increased approximately two-fold by perfusion with paraquat in medium containing glucose and three-fold in the absence of glucose.

Calcium absorption during development: experimental studies of the rat small intestine.

During the period of natural weaning in the rat (beginning during the third week) the mechanisms of calcium absorption from the duodenum change from an efficient nonsaturable process, which is insensitive to vitamin D, to a combination of a less efficient nonsaturable process and a saturable vitamin D-dependent component. The stimulatory effect of vitamin D on active calcium transport requires the development of mucosal receptors for 1,25-(OH)2D3 and increasing circulating levels of 1,25-(OH)2D3. Concurrent increases in the concentration of the cytosolic calcium-binding protein (CaBP) support the suggestion that the CaBP mediates, in part, the effect of 1,25-(OH)2D3 on active calcium transport. The decline in the efficiency of nonsaturable calcium absorption during the third week coincides with, but is probably not due to, the loss of pinocytotic capacity of the small intestine. The time of appearance of the receptors for 1,25-(OH)2D3 and the decline in nonsaturable calcium absorption are under glucocorticoid control as are several other maturational events in the intestine at this time.