- Email: [email protected]
Effect of nicotine and cotinine on perinatal rat brain aromatase mRNA expression and activity
208
Poster Session 4C. Reproductive Toxicology
generation of the zona fasciculata cells. These findings suggest that precision-cut adrenal tissue-slice culture is a useful test system to examine metabolism-dependent toxicity in humans and wild species.
P4C. Reproductive Toxicology
iP4C27 I COMPARATIVE PHARMACOKINETICS AND METABOLISM OF ETHYLENE GLYCOL IN PREGNANT RATSAND RABBITS
E.W. Carney *, L.H. Pottenger, MJ. Bartels. R. Jackh 1 • J.E Quast.
Toxicology ResearchLaboratory, The Dow Chemical Co., Midland, MI, USA; J BASF AG, Ludwigshafen, Germany In rats and mice, large oral bolus doses of ethylene glycol (EG) cause malformations, which are thought to be due to an acidic metabolite, glycolic acid (GA). In contrast, EG is not teratogenic in rabbits at doses up to 2000 mg/kg/day. This study compared the pharmacokinetics ofEG and metabolites in pregnant rats and rabbits, as a first step in determining which of these species best represents humans. Initially, we characterized the maternal blood time courses for EG, GA and oxalic acid (OX), as well as acid-base balance parameters, following gavage administration of 2500 mg/kg of EG to arterial-cannulated, gestation day (gd) 9 rabbits. Similar to prior rat data, peak EG levels in rabbits occurred I h after dosing, but in contrast to rats, rabbits exhibited a flat GA blood conce ntration curve (maxim um levels extended from 4-12 h post-dosing ) and no evidence of metabolic acidosis . Subsequently we conducted a direct species compariso n ofEG, GA and OX levels in maternal blood and extraembryonic fluids (EEF) of gd 10 rats and gd 9 rabbits at selected times after dosing with 500 or 2500 mg/kg of EG. Key findings were: (I ) maternal blood concentrations of EG were comparable in rats and rabbits : (2) levels of GA in maternal blood and EEF were 3 to 36-fold lower in rabbits than in rats; (3) in rats, concentrations of GA were higher in EEF than in maternal blood ; (4) in rabbits, GA was generally lower in EEF than in maternal blood. In both species, OX was a minor metabolite of EG, with levels in maternal blood and EEF samples generally near or below background . These data provide further support for the hypothesis that GA is the ultimate developmenta l toxicant in rats, and suggest that the lack of teratogenicity in the rabbit is due to decreased embryonic exposure to GA, either because of lower maternal GA production, enhanced GA elimination, and/or decreased transfer of GA across the visceral yolk sac to the embryo. Sponsored by CEFIC (European Chemical Industry Council) and CMA (U.S. Chemical Manufactu rers Association).
IP4C28 ! EFFECTOF NICOTINE AND COTININE ON PERINATAL RATBRAIN AROMATASE mRNA EXPRESSION AND ACTIVITY
A. Sarasin, M.E. Lauber, W. Lichtensteiger
*. Instituteof
Pharmacology; University of Zurich, CH-8057 ZUrich, Switzerland One week expos ure of rat fetuses to nicotine interferes with sexual brain differentiation as indicated in adult male offspring by female-type sweet preference and reduced male sexual behavior (Lichtensteiger and Schlumpf, 1985; Segarra and Strand , 1989). The drug abolishes the fetal (gestational day (GD) 18) testosterone peak in males and a sexual dimorphi sm in the activity of brain arornatase (CYPI9), the enzyme converting testosterone to estradiol , on postnatal day (PN) 6 (von Ziegler et al., 1991). Possible effects on brain aromatase were further investigated in fetuses and offspring of time-preg nant Long Evans rats. Early postnatal aromatase mRNA expression, analyzed by in situ hybridization with two 33P-labeled antisense oligonucleotides complementary to 5'- and 3' -regions of
rat aromatase cDNA and quantitative image analysis, is sexually dimorphic (Lauber et al., Neuroendocrinology 66, 173, 1997). Prenatal nicotine exposure did not affect sex differences in regional brain aromatase mRNA levels. Effects of nicotine and its metabolite cotinine on the activity of fetal and neonatal brain aromatase were studied in vitro in a homogenate containing the main perinatal expression sites, preoptic area, bed nucleus of stria terminalis and medial amygdala, with [1t3-3H]androstenedione as substrate. Both compounds inhibited aromatase activity competitively. Ki values (nicotine 443 j.tM at GD19, 434 /LM at PN2; cotinine 2 14 /LM at GDI9) are somewhat higher than previously reported for aromatase of human placental microsomes (Barbieri et al., 1986). In order to assess the significance of direct enzyme inhibition for in vivo conditions, nicotine and cotinine concentrations in rat maternal and fetal plasma and fetal brain at developmentally active nicotine dosages are presently being analyzed by gas chromatography-mass spectrometry.
IP4C30 I PATERNAL EXPOSURE TO CYCLOPHOSPHAMIDE (CPA)AFFECTSDNA DAMAGE RESPONSEJREPAIR GENEEXPRESSION IN RAT EMBRYOS
W. Harrouk *, B. Robaire, B.F. Hales. DepartmentofPharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada Chronic administration of CPA, an anticancer alkylating agent, to male rats results in up to 90% peri-implantatio n progeny loss. CPA treatment has a major effect on spermatid s, ceUs in which DNA is tightly packed. We hypothesize that the fate of embryos sired by CPA-treated males depends on the ability of the pre-implantation embryo to respond to DNA damage and repair it. To test this hypothesis , we assessed the expression of three DNA damage responselre pair genes in morulae and blastocysts sired by male rats treated for 4 weeks with either saline or 6 mg/kg/day CPA. Antisense RNA, synthesized from polyA mRNA, was used to probe blots with the cDNAs for p2 1cipllwafl, a cell cycle checkpoint gene; apurinic/apyrimidinic endonuclease/redox factor-I (APE/ref-I) , a multifunctional DNA repair and stress response gene; and PMS2, a DNA mismatch repair gene. All three genes were expressed in control morulae and blastocysts to a similar extent. No differences in expressio n were found between control and CPA-sired morulae. In contrast, a dramatic 5- 12-fold decrease in the expression of all three genes (p21Cipliwafl , P :s 0.007 ; APElref-l , P s 0.01; PMS2 , P :s 0.05) was observed in blastocysts sired by CPA-treated males compared to control. Thus, fertilization by CPA damaged spermatozoa led to a stage-specific, dramatic decrease in the transcripts for certain DNA damage response/ repair genes in pre-implantation embryos. A deficiency in these gene products may contribute to early embryo death in the progeny of CPA-exposed males. Supported by the Medical Research Council of Canada.
IP4C31 I A TERATOGEN INHIBITINGCHOLESTEROL
BIOSYNTHESIS PHENOCOPIES SONIC HEDGEHOG DEVELOPMENTAL GENEMUTATION IN RAT EMBRYOS
M. Kolf-Clauw *1 , F. Clotmarr' . C. Roux 2 , J.J. Picard3 . I Toxicology
Dept., Alfort VeterinarySchool, 94704 Maisons-Alfort; Embryology, CHU St Antoine, 75012 Paris, France; 3 Developmental Genetics, Place Croix du Sud 5. UCL, 1348 Louvain-La-Neuve, Belgium 2 Pathological
Inhibition of the last step of cholesterol biosynthesis in rat causes craniofacial and cerebral defects related to holoprosencephaly (HPE) . These defects resemble the holoprosencephalic phenotype of embryos lacking a functional Shh gene (Sonic Hedgehog). This patterning gene encodes for a cholestero l-dependent signaling protein. To test the hypothesis that a HPE-inducin g teratogen inhibiting 7-dehy-
Poster Session 4C. Reproductive Toxicology
generation of the zona fasciculata cells. These findings suggest that precision-cut adrenal tissue-slice culture is a useful test system to examine metabolism-dependent toxicity in humans and wild species.
P4C. Reproductive Toxicology
iP4C27 I COMPARATIVE PHARMACOKINETICS AND METABOLISM OF ETHYLENE GLYCOL IN PREGNANT RATSAND RABBITS
E.W. Carney *, L.H. Pottenger, MJ. Bartels. R. Jackh 1 • J.E Quast.
Toxicology ResearchLaboratory, The Dow Chemical Co., Midland, MI, USA; J BASF AG, Ludwigshafen, Germany In rats and mice, large oral bolus doses of ethylene glycol (EG) cause malformations, which are thought to be due to an acidic metabolite, glycolic acid (GA). In contrast, EG is not teratogenic in rabbits at doses up to 2000 mg/kg/day. This study compared the pharmacokinetics ofEG and metabolites in pregnant rats and rabbits, as a first step in determining which of these species best represents humans. Initially, we characterized the maternal blood time courses for EG, GA and oxalic acid (OX), as well as acid-base balance parameters, following gavage administration of 2500 mg/kg of EG to arterial-cannulated, gestation day (gd) 9 rabbits. Similar to prior rat data, peak EG levels in rabbits occurred I h after dosing, but in contrast to rats, rabbits exhibited a flat GA blood conce ntration curve (maxim um levels extended from 4-12 h post-dosing ) and no evidence of metabolic acidosis . Subsequently we conducted a direct species compariso n ofEG, GA and OX levels in maternal blood and extraembryonic fluids (EEF) of gd 10 rats and gd 9 rabbits at selected times after dosing with 500 or 2500 mg/kg of EG. Key findings were: (I ) maternal blood concentrations of EG were comparable in rats and rabbits : (2) levels of GA in maternal blood and EEF were 3 to 36-fold lower in rabbits than in rats; (3) in rats, concentrations of GA were higher in EEF than in maternal blood ; (4) in rabbits, GA was generally lower in EEF than in maternal blood. In both species, OX was a minor metabolite of EG, with levels in maternal blood and EEF samples generally near or below background . These data provide further support for the hypothesis that GA is the ultimate developmenta l toxicant in rats, and suggest that the lack of teratogenicity in the rabbit is due to decreased embryonic exposure to GA, either because of lower maternal GA production, enhanced GA elimination, and/or decreased transfer of GA across the visceral yolk sac to the embryo. Sponsored by CEFIC (European Chemical Industry Council) and CMA (U.S. Chemical Manufactu rers Association).
IP4C28 ! EFFECTOF NICOTINE AND COTININE ON PERINATAL RATBRAIN AROMATASE mRNA EXPRESSION AND ACTIVITY
A. Sarasin, M.E. Lauber, W. Lichtensteiger
*. Instituteof
Pharmacology; University of Zurich, CH-8057 ZUrich, Switzerland One week expos ure of rat fetuses to nicotine interferes with sexual brain differentiation as indicated in adult male offspring by female-type sweet preference and reduced male sexual behavior (Lichtensteiger and Schlumpf, 1985; Segarra and Strand , 1989). The drug abolishes the fetal (gestational day (GD) 18) testosterone peak in males and a sexual dimorphi sm in the activity of brain arornatase (CYPI9), the enzyme converting testosterone to estradiol , on postnatal day (PN) 6 (von Ziegler et al., 1991). Possible effects on brain aromatase were further investigated in fetuses and offspring of time-preg nant Long Evans rats. Early postnatal aromatase mRNA expression, analyzed by in situ hybridization with two 33P-labeled antisense oligonucleotides complementary to 5'- and 3' -regions of
rat aromatase cDNA and quantitative image analysis, is sexually dimorphic (Lauber et al., Neuroendocrinology 66, 173, 1997). Prenatal nicotine exposure did not affect sex differences in regional brain aromatase mRNA levels. Effects of nicotine and its metabolite cotinine on the activity of fetal and neonatal brain aromatase were studied in vitro in a homogenate containing the main perinatal expression sites, preoptic area, bed nucleus of stria terminalis and medial amygdala, with [1t3-3H]androstenedione as substrate. Both compounds inhibited aromatase activity competitively. Ki values (nicotine 443 j.tM at GD19, 434 /LM at PN2; cotinine 2 14 /LM at GDI9) are somewhat higher than previously reported for aromatase of human placental microsomes (Barbieri et al., 1986). In order to assess the significance of direct enzyme inhibition for in vivo conditions, nicotine and cotinine concentrations in rat maternal and fetal plasma and fetal brain at developmentally active nicotine dosages are presently being analyzed by gas chromatography-mass spectrometry.
IP4C30 I PATERNAL EXPOSURE TO CYCLOPHOSPHAMIDE (CPA)AFFECTSDNA DAMAGE RESPONSEJREPAIR GENEEXPRESSION IN RAT EMBRYOS
W. Harrouk *, B. Robaire, B.F. Hales. DepartmentofPharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada Chronic administration of CPA, an anticancer alkylating agent, to male rats results in up to 90% peri-implantatio n progeny loss. CPA treatment has a major effect on spermatid s, ceUs in which DNA is tightly packed. We hypothesize that the fate of embryos sired by CPA-treated males depends on the ability of the pre-implantation embryo to respond to DNA damage and repair it. To test this hypothesis , we assessed the expression of three DNA damage responselre pair genes in morulae and blastocysts sired by male rats treated for 4 weeks with either saline or 6 mg/kg/day CPA. Antisense RNA, synthesized from polyA mRNA, was used to probe blots with the cDNAs for p2 1cipllwafl, a cell cycle checkpoint gene; apurinic/apyrimidinic endonuclease/redox factor-I (APE/ref-I) , a multifunctional DNA repair and stress response gene; and PMS2, a DNA mismatch repair gene. All three genes were expressed in control morulae and blastocysts to a similar extent. No differences in expressio n were found between control and CPA-sired morulae. In contrast, a dramatic 5- 12-fold decrease in the expression of all three genes (p21Cipliwafl , P :s 0.007 ; APElref-l , P s 0.01; PMS2 , P :s 0.05) was observed in blastocysts sired by CPA-treated males compared to control. Thus, fertilization by CPA damaged spermatozoa led to a stage-specific, dramatic decrease in the transcripts for certain DNA damage response/ repair genes in pre-implantation embryos. A deficiency in these gene products may contribute to early embryo death in the progeny of CPA-exposed males. Supported by the Medical Research Council of Canada.
IP4C31 I A TERATOGEN INHIBITINGCHOLESTEROL
BIOSYNTHESIS PHENOCOPIES SONIC HEDGEHOG DEVELOPMENTAL GENEMUTATION IN RAT EMBRYOS
M. Kolf-Clauw *1 , F. Clotmarr' . C. Roux 2 , J.J. Picard3 . I Toxicology
Dept., Alfort VeterinarySchool, 94704 Maisons-Alfort; Embryology, CHU St Antoine, 75012 Paris, France; 3 Developmental Genetics, Place Croix du Sud 5. UCL, 1348 Louvain-La-Neuve, Belgium 2 Pathological
Inhibition of the last step of cholesterol biosynthesis in rat causes craniofacial and cerebral defects related to holoprosencephaly (HPE) . These defects resemble the holoprosencephalic phenotype of embryos lacking a functional Shh gene (Sonic Hedgehog). This patterning gene encodes for a cholestero l-dependent signaling protein. To test the hypothesis that a HPE-inducin g teratogen inhibiting 7-dehy-