Immature Male Wistar Rats Research Articles

Toxicity of 3,3',4,4'- and 2,2',5,5'-tetrabromobiphenyl: correlation of activity with aryl hydrocarbon hydroxylase induction and lack of protection by antioxidants.

3,3',4,4'-Tetrabromobiphenyl is a minor component of commercial polybrominated biphenyl (PBB) mixture fireMaster BP-6 and is a potent inducer of aryl hydrocarbon hydroxylase (AHH). A single ip dose of 3,3',4,4'-tetrabromobiphenyl (150 mumol/kg) caused significant reduction in the growth rate in the immature male Wistar rat, as well as pale enlarged livers and marked reduction in thymus size. Under light microscopy, hepatocytes were enlarged and vacuolated. The vacuoles, which were most prominent in the midzonal region of the lobule, corresponded to fat droplets in oil-red-O-stained sections. The thymus, especially the cortex, was markedly depleted of lymphocytes. Neither the reduced growth, altered organ weights nor the histopathology was reversed for the duration of the study by the coadministration of the antioxidants butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), or vitamin E. Vitamin E did, however counter the negative effect of 3,3',4,4'-tetrabromobiphenyl on growth during the first 5 d of the study. 2,2',5,5'-Tetrabromobiphenyl, also a minor component of fireMaster BP-6, is a weak phenobarbital-type inducer of cytochrome P-450. When administered at the same dose, 2,2',5,5'-tetrabromobiphenyl did not elicit any observed toxic effects. These data confirm the correlation between AHH induction and toxicity for these PBBs and suggest that 3,3',4,4'-tetrabromobiphenyl may significantly contribute to the toxicity of fireMaster BP-6. Although there is evidence that polychlorinated biphenyls, and perhaps 2,3,7,8-tetrachlorodibenzo-p-dioxin, exert certain toxic effects via a lipid peroxidation mechanism, the toxic changes measured during this study were not reversed by the administration of the antioxidants.

The effects of twenty-two organochlorine pesticides as inducers of the hepatic drug-metabolizing enzymes

1. 1. The effects of 22 organohalogen pesticides as inducers of hepatic drug-metabolizing enzymes in the immature male Wistar rat have been determined, this group includes four isomeric hexachlorocyclohexanes, technical chlordane, α-chlordane, γ-chlordane, oxychlordane, trans-nonachlor, heptachlor, heptachlor epoxide, aldrin, dieldrin, kepone, toxaphene, mirex, hexachlorobenzene (HCB) and several DDT analogs. 2. 2. With the exception of HCB, all of the pesticides induced microsomal dimethylaminoantipyrine, N-demethylase and aldrin epoxidase activities and the cytochrome P-450 content of microsomes from animals pretreated with most of the compounds was also increased compared to control rats. These pesticides all resembled phenobarbitone in their mode of induction. 3. 3. The effects of HCB as a microsomal enzyme inducer resembled those observed after coadministration of phenobarbitone plus 3-methylcholanthrene.

Polybrominated biphenyls as aryl hydrocarbon hydroxylase inducers: structure-activity correlations

The synthesis of all possible laterally-substituted polybrominated biphenyl (PBB) congeners containing two para bromines is described. Using enzymic, electrophoretic and ligand-binding assays that distinguish between phenobarbitone(PB)- and 3-methylcholanthrene(MC)-type inducers, the synthetic PBBs were evaluated as inducers of liver microsomal drug-metabolizing enzymes in the immature male Wistar rat. 4,4′-Dibromobiphenyl resembled PB in its mode of induction whereas all the meta-brominated derivatives of 4,4′-dibromobiphenyl, namely 3,4,4′-tri-, 3,4,4′,5-tetra-, 3,3′, 4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexabromobiphenyl, resembled MC in their mode of induction. The results obtained with 3,4,4′-tribromobiphenyl demonstrate that, in contrast to the polychlorinated biphenyls (PCBs), a single meta halogen substituent is sufficient to abolish the PB-type characteristics of 4,4′-dibromobiphenyl and convert it to a strictly MC-type inducer. PBBs which induce AHH activity must be substituted at both para positions and at one, two, three or four meta positions. Ortho-substitution of PBBs which contain only lateral bromine groups may also give compounds which are aryl hydrocarbon hydroxylase (AHH) inducers. One of the MC-type PBBs, namely 3,3′,4,4′-tetrabromobiphenyl, which has been tentatively identified in the commercial PBB mixture, fireMaster BP-6, was at least 50 times more potentias an inducer of AHH activity than the commercial PBB mixture. The induction of AHH by 3,3′,4,4′-tetrabromobiphenyl was accompanied by a dose-dependent decrease in both thymus and spleen weights. The thymus and/or spleen weights were decreased in rats treated with the other MC-type PBBs which further supports the correlation between the toxicity of the PBBs and their ability to induce AHH.

PCB isomers and congeners: Induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase enzyme activities in rat hepatoma cells

The effects of structure on the activity of 15 polychlorinated biphenyl (PCB) isomers and congeners as inducers of cytochrome P-448-dependent monooxygenases in rat hepatoma cell cultures was investigated. All the PCBs which have previously been classified as aryl hydrocarbon hydroxylase (AHH) inducers in the immature male Wistar rat also induced benzo[a]pyrene hydroxylase and ethoxyresorufin (ER) O-deethylase activity in the rat hepatoma H-4-II-E cells. The relative activities of the compounds were evaluated by comparing the doses required to half-maximally induce the two enzyme activities. The most potent PCB inducer, 3,3' ,4,4' ,5-pentachlorobiphenyl, exhibited molar EC 50 values of 2.48 · 10 −10 and 2.40 · 10 −10 for the induction of ER O-deethylase and benzo[a]pyrene hydroxylase enzyme activities, respectively. These values were only 3- to 4-fold lower than the data obtained for the highly toxic 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The relative activities of the PCB congeners as inducers of cytochrome P-448-dependent monooxygenase in vitro were similar to their potencies as microsomal enzyme inducers in the immature male rat.

Halogenated biphenyls as AHH inducers: Effects of different halogen substituents

4′-Iodo-, 4′-bromo-, 4′-chloro- and 4′-fluoro-2,3,4,5-tetrachlorobiphenyl were administered to immature male Wistar rats and the effects of this homologous series of 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal drug-metabolizing enzymes were determined. All the halogenated biphenyls increased microsomal benzo[a]pyrene hydroxylase (or aryl hydrocarbon hydroxylase, AHH), ethoxyresorufin (ER) O-deethylase and dimethylaminoantipyrine (DMAP) N-demethylase. The effects of the 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal enzyme activities and on the relative peak intensities and spectral shifts of the reduced cytochrome P-450:CO and ethylisocyanide (EIC) binding difference spectra were similar to those observed after coadministration of phenobarbitone (PB) and 3-methylcholanthrene (MC). The relative activities of the halogenated biphenyls were determined using two in vitro assays; namely cytochrome P-448 associated induction in rat hepatoma H-4-II E cells in culture and competitive binding to the hepatic cytosolic Ah receptor protein from male Wistar rats. Dose-response experiments for the iodo, bromo, chloro and fluoro analogs gave EC 50(M) values of 8.5×10 −9, 6.6×10 −8, 5.7×10 −7, and 3.3×10 −5, and 1.5×10 −6, 2.5×10 −6, 4.1×10 −6 and 2.5×10 −5 for the ER O-deethylase induction and receptor binding assays respectively. The relative potencies of the 4′-halo-2,3,4,5-tetrachlorobiphenyls followed the order I>Br>Cl>F for both assays and differences in the EC 50 values for the iodo and fluoro analogs were greater than three orders of magnitude for ER O-deethylase induction in rat hepatoma cells in culture. One possible explanation for these effects may be associated with differences in the polarizability of the laterally substituted halogen groups. However, other differences in the physico-chemical properties of the halogen atoms may also be important.

Potent induction of rat liver microsomal, drug-metabolizing enzymes by 2,3,3′,4,4′,5-hexabromobiphenyl, a component of fireMaster

The multistep synthesis and purification of 2,3,3′,4,4′,5-hexabromobiphenyl (HBBp) is described. Capillary gas chromatography revealed that HBBp comprises 0.05% of the industrial polybrominated biphenyl (PBB) mixture, fireMaster BP-6 (lot 7062). When administered to immature male Wistar rats, HBBp caused a dose-dependent increase in (a) the activity of benzo[ a]pyrene (B[ a]P) hydroxylase (AHH) and 4-chlorobiphenyl (4-CBP) hydroxylase and (b) the concentration of cytochrome P-450. Sodium dodecyl sulfate (SDS)-gel electrophoresis indicated that these increases in cytochrome P-450 and cytochrome P-450-dependent monooxygenase activities were accompanied by a dose-dependent intensification of a protein of relative molecular weight (Mr) 55 000 which comigrated with the major 3-methylcholanthrene(MC)-inducible form of cytochrome P-450 (i.e., cytochrome P-448). Like MC, but in contrast to phenobarbitone (PB), HBBp competitively displaced 2,3,7,8-[ 3H]tetrachlorodibenzo- p-dioxin ([ 3H]-TCDD) from the cytosolic protein thought to be the receptor for cytochrome P-448 induction. The results indicate that HBBp is a potent inducer of cytochrome P-448 and as such is the third MC-type inducer identified in fireMaster BP-6.

Octachloronaphthalene induction of hepatic microsomal aryl hydrocarbon hydroxylase activity in the immature male rat

Administration of octachloronaphthalene to immature male Wistar rats resulted in a dose-dependent increase in several enzymic, electrophoretic and spectral parameters associated with induction of the hepatic microsomal enzymes. Compared to corn-oil (control) treated animals octachloronaphthalene (150 μmol · kg −1 induced hepatic cytochrome P-450 (1.5-fold), benzo [ a]pyrene hydroxylase (18-fold) and 4-chlorobiphenyl hydroxylase (18-fold) enzyme activities. In addition to increases in the relative peak intensities of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide (EIC) difference spectra the peak maxima were observed at 448.5 and 452.2/428.0 nm, respectively. The effects of administering octachloronaphthalene to the rat were similar to those observed after pretreatment with 3-methylcholanthrene (MC) and electrophoresis of the induced microsomal proteins showed that both compounds enhanced heme-staining peptides with comparable electrophoretic mobilities. Moreover coadministration of MC (3 × 10 βmol · kg −1) and octachloronaphthalene (2 × 150 μmol · kg −1) indicated that their inductive effects were not additive. It was concluded that octachloronaphthalene was an MC-type inducer of hepatic microsomal enzymes.

Reversal by arginine-vasotocin of the effects of pinealectomy on the amount of 5-hydroxytryptamine in the hypothalamus and the concentrations of luteinizing hormone and follicle-stimulating hormone in the plasma of immature male rats.

Pinealectomy in immature (25-day-old) male Wistar rats significantly decreased the content of 5-hydroxytryptamine (5-HT) in the hypothalamus and increased concentrations of plasma LH and FSH 3 days after surgery. Extremely small amounts (10(-4) pg) of arginine-vasotocin (AVT) injected into the third ventricle (pineal recess) 5 min after pinealectomy completely prevented the decrease in the hypothalamic content of 5-HT and the increase in the concentration of LH and FSH in the plasma. It is suggested that AVT prevented the increase of plasma levels of LH and FSH after pinealectomy by increasing levels of 5-HT at postsynaptic receptor sites.

OESTROGEN INDUCTION OF THYMIDINE KINASE IN THE PITUITARY OF THE MALE RAT: CORRELATION BETWEEN INDUCER ABILITY AND OESTROGENIC POTENCY

Laboratoire de Neurobiologie Moléculaire (Equipe de Recherche associée au C.N.R.S. no. 567), Avenue du Général Ledere, 35031 Rennes Cedex, France (Received 29 December 1975) A single injection of oestradiol (OE2) to intact immature male rats induces pituitary thymidine kinase activity and DNA biosynthesis (Valotaire, 1975; Valotaire, Le Guellec, Kercret, Guellaën & Duval, 1975). The minimum active dose of the oestrogenic compound is 20 ng/rat; a dose which does not affect peripheral levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (de Jong, Uilenbroek & van der Molen, 1975; Valotaire et al. 1975). It appeared interesting to determine if only oestrogenic substances could produce such an induction and if this effect was correlated with an uptake of the hormone by the pituitary. Immature male Wistar rats (42-43 days old) were injected s.c., twice at 24 h intervals, with 0 (control), 1 μg OE2, oestradiol-17α (αOE2), oestrone (OE1), oestriol (OE3), diethylstilboestrol (DES) or

Gonadal effects of vasectomy and vasoligation.

During a 28-week study, vasectomy and vasoligation of immature male Wistar rats revealed that there was a significant decrease in urinary 17-ketosteroid in the vasectomized group at week 15; at week 28 there were significant decreases in the weights of the testes of the test groups, as compared to those receiving sham operations, with maximum alterations in the vasectomized rats. Small, soft discolored testes with cysts in the cauda epididymis and vas deferens regions occurred frequently in the test groups. The output of 17-ketosteroid in the urine and the findings in the testes indicate significant alterations in the morphology and function of the testes and suggest the need for caution and extensive investigations in man before recommending vasectomy as a simple, innocuous, "physiologic" means to ensure conception control.

II. Effects of Vitamin D Deficiency

Introduction The present report deals with the effects of vitamin D deficiency on the dynamics of chemical growth and composition during the first 75 days of. life in the immature male Wistar rat. In the normal animal this early period of life is characterized by specific changes in chemical composition of bone, and these changes have been correlated with chemical growth and turnover and linear growth in other publications in this series.* Many studies have been reported which deal with the effects of vitamin D deficiency on somatic and skeletal growth,† skeletal morphology and maturation,‡ and on chemical composition and turnover of bone.§ The following studies were undertaken to correlate chemical growth and turnover of the entire skeleton with serial changes in the chemical composition and linear growth of long bones in animals maintained on vitamin D-deficient and normal diets. Materials and Methods Male Wistar rats were used throughout this

Chemical growth dynamics of the skeleton in the immature rat. III. Correlation of growth and morphology of long bones with chemical growth in normal and vitamin D-deficient animals.

Introduction This publication presents data pertaining to linear bone growth and morphology in immature male Wistar rats on normal and vitamin D-deficient diets and correlates these data with chemical growth and turnover of the skeleton, previously reported.* Studies involving linear growth, histology, and chemistry of long bones have long been accepted individually as representative indices of the influences of metabolic and endocrine factors upon the skeleton.† However, there has been a paucity of studies correlating these parameters, particularly as they relate to total skeletal chemistry and turnover. The present series of studies were undertaken to provide a correlated chemical, radiological, and morphological interpretation of skeletal growth from birth through 75 days of age in male Wistar rats maintained on normal and vitamin D-deficient diets. Furthermore, these studies bring to focus the observations of earlier workers suggesting that the chemical changes induced in bone by a vitamin D-deficient diet may be