Evidence for increased formation of preprothrombin and the noninvolvement of vitamin K-dependent reactions in sex-linked hyperprothrombinemia in the rat.

The rate of appearance of plasma prothrombin was measured in vitamin K-deficient male and female rats after the administration of vitamin K₁, and the disappearance of prothrombin was measured in normal rats after injection of cycloheximide. The results suggest that hyperprothrombinemia in female rats is due to a faster rate of formation of the clotting protein rather than to a slower rate of its degradation. Preprothrombin activity in liver microsomes was higher in warfarin-treated female rats than in warfarintreated male rats; but the activity of preprothrombin in liver disappeared at approximately the same rate in both sexes after administration of vitamin K. The rate and extent of vitamin K-dependent formation of γ-carboxyglutamic acid and the appearance of prothrombin activity in vitro were not significantly different between the sexes. These results suggest that elevated levels of plasma prothrombin in female rats are probably due to a higher rate of synthesis of preprothrombin and not to any difference in the vitamin K-dependent step. A difference was observed in the amount of cycloheximide required to inhibit synthesis of liver microsomal protein in the two sexes.     

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.     

Seasonal change and sex difference in drug-metabolizing activity in frog liver microsomes

The drug-metabolizing activities (aminopyrine N-demethylase and p-nitroanisole O-demethylase activities) have been measured at monthly intervals throughout the year in liver microsomes of male and female Japanese bullfrogs, Rana catesbeiana. The aminopyrine N-demethylase activity based on cytochrome P-450 of both sexes was significantly higher in May-July (spring-summer) than in August-October (summer-autumn). On the contrary, the p-nitroanisole O-demethylase activity based on cytochrome P-450 of males was significantly higher in July-October (summer-autumn) than in May-June (spring). However, there was no seasonal changes in the O-demethylase activity in females. There were significant differences between males and females in the N-demethylase activity in August-October and in the O-demethylase activity in May-June (or July-October).     

UDP-galactose:globoside galactosyltransferase in murine kidney

There are increased levels of stage-specific embryonic antigens-3 and -1 (SSEA-3 and SSEA-1) globo-series glycolipids in male versus female DBA/2 and C57BL/6 kidneys, respectively. To determine what enzymatic steps may be responsible for these differences, the activity and properties of UDP-galactose:globoside galactosyltransferase were studied in male and female mouse kidney microsomes. This enzyme participates in the biosynthesis of galactosylgloboside, SSEA-3 glycolipid; the reaction product was identified by high performance thin-layer chromatography (HPTLC) immunostaining. In C57BL/6 mice, the specific activity of the enzyme, in the presence of CHAPS, was 2-fold greater in the male than that in the female. Optimum pH for the enzyme from both sexes was about 5.6, and Mn2+ was essential for maximal activity. Fifty percent of the male and female enzyme activity was lost after preincubating the microsomes for 1 min at 55 degrees C; thereafter, the enzyme from female microsomes had a slower rate of denaturation. The Km for globoside in presence of sodium cholate for both male and female was 0.035 mM, but it was approximately 2-fold greater for the female in presence of CHAPS. The enzyme in male and female microsomes was differentially activated by CHAPS and cholate. The results suggest the presence of an enzyme modulator in these membranes. In DBA/2 mice, the enzyme activity was about 2-fold greater in males than that in the female. The specific activity of the enzyme in the two strains was of a similar magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450 difference spectra of microsomes from several insecticide-resistant and -susceptible strains of the housefly, Musca domestica L

Cytochrome P-450 difference spectra were obtained with microsomes prepared from several strains of the housefly, Musca domestica, that were susceptible or resistant to insecticides. Based on Type I, Type II and Type III spectral interactions, both quantitative and qualitative variations are apparent among strains. Cytochrome P-450 from Fc and dimethoate strains had spectral characteristics which were most different from those of susceptible strains. No correlation could be made between strains having known high mixed-function oxidase activity and any single cytochrome P-450 spectral characteristic.     

Biotransformation in Egyptian spiny mouse Acomys cahirinus

The activities of several representative biotransformation enzymes were determined in male and female spiny mouse tissues. Cytochrome P450 monooxygenase activity toward benzo(a)pyrene was significantly greater in female spiny mouse intestine than in males. Activity toward benzphetamine in both sexes was high in the liver, with little activity in the kidney and intestine. Sulfotransferase activity was high in kidney and intestine of female spiny mice but undetectable in the same tissues in males. Hepatic glutathione s-transferase activity towards 1-chloro-2,4-dinitrobenzene in females was significantly higher than in males. UDP-Glucuronosyltransferase activity toward 1-naphthol in both sexes in the kidney was significantly higher than hepatic and intestinal activity. Intestinal N-acetyltransferase activity towards 2-aminofluorene and β-naphthylamine was significantly greater in females than males. No consistent relation appeared to exist between biotransformation activities in spiny mouse and those in other related rodent species.     

Effects of emulsified perfluorochemicals on liver aryl esterase in rats.

1. The effects of intravenous (i.v.) injection of various perfluorochemical (PFC) emulsions have been studied separately in male and female rats. 2. Injection of 10 ml/kg body weight of either Fluosol-DA 20% (F-DA) or a novel perfluorodecalin emulsion containing a C-16 oil additive in male rats increased liver weight up to 7 days later; no corresponding change occurred in response to injection of Oxypherol (FC-43). 3. Liver weight was also increased in female rats at 72 hr after injection of the novel emulsion but this was less pronounced than in males; liver weight in female rats was unchanged in response to injection of either F-DA or FC-43. 4. Mean liver aryl esterase activity in male rats was increased 2- to 3-fold (P less than 0.05) at 7 days after injection of the novel emulsion. No significant alterations in aryl esterase activity occurred in response to injection of either F-DA or FC-43, although in both cases there was a trend towards increased activity. 5. Liver aryl esterase activity in female rats was significantly (P less than 0.05) decreased at 72 hr following FC-43 injection with similar, but much less pronounced, changes occurring in response to injection of F-DA and the novel emulsion. 6. These results show that injection of a single low dose of emulsified PFCs into rats can alter hepatic microsomal aryl esterase activity but the response is highly variable, depending on composition of emulsion injected and sex of recipient.     

Egasyn, a protein which determines the subcellular distribution of beta-glucuronidase, has esterase activity

The glycoprotein egasyn complexes with and stabilizes precursor beta-glucuronidase in microsomes of several mouse organs. Several observations indicate egasyn is, in addition, an esterase. Liver homogenates of egasyn-positive strains have specific electrophoretically separable esterases which are absent in egasyn-negative mice. These esterases react with anti-egasyn serum. A specific esterase was likewise complexed with immunopurified microsomal beta-glucuronidase. The esterases were, like egasyn and microsomal beta-glucuronidase, concentrated in the microsomal subcellular fraction. Egasyn which is not bound to beta-glucuronidase, which represents 80-90% of total liver egasyn, is not complexed with other liver proteins. Egasyn, therefore, specifically stabilizes beta-glucuronidase in microsomes. The esterase activity is inhibited by bis-p-nitrophenyl phosphate indicating it is a carboxyl esterase. Several possible functions of egasyn-esterase activity are discussed.     

Effects of TCDD upon IkappaB and IKK subunits localized in microsomes by proteomics

Biochemical studies have shown that microsomes represent an important subcellular fraction for determining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects. Proteomic analysis by two-dimensional gel-mass spectrometry of liver microsomes was undertaken to gain new insight into the actions of TCDD in male and female rats. Proteomic analysis showed TCDD induced several xenobiotic metabolism enzymes as well as a protein at 90kDa identified by mass spectrometry as IkappaB kinase beta/IKK2. This observation led to the discovery of other NF-kappaB binding proteins and kinases in microsomes and effects by TCDD. Western blotting for IKK and IkappaB family members in microsomes showed a distinct pattern from cytosol. IKK1 and IKK2 were both present in microsomes and were catalytically active although, unlike cytosol, IKKgamma/NEMO was not detectable. TCDD exposure produced an elevation in cytosolic and microsomal IKK activity of both genders. The NF-kappaB binding proteins IkappaBbeta and IkappaBgamma were prevalent in microsomes, while IkappaBalpha and IkappaB epsilon proteins were absent. TCDD treatment produced hyperphosphorylation of microsomal IkappaBbeta in both sexes with females being most sensitive. In cytosol, IkappaBalpha, IkappaBbeta, and IkappaB epsilon, but not IkappaBgamma, were clearly observed but were not changed by TCDD. Overall, proteomic analysis indicated the presence of NF-kappaB pathway members in microsomes, selectively altered by dioxin, which may influence immune and inflammatory responses within the liver.     

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Hepatic Distribution of Iron,Copper, Zinc,and Magnesium in Rats

The distribution of iron, copper, zinc, and magnesium in hepatic subcellular fractions of male and female rats treated with 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was determined. Animals received 40 μg TCDD per kilogram per day for three days by mouth (PO) or the vehicle and were killed seven or nine days posttreatment. Iron, copper, zinc, and magnesium were determined by atomic absorption spectroscopy. The iron content of liver from female animals was twofold higher than male animals. The administration of TCDD increased the iron content of mitochondria in female and male rats and decreased iron content of microsomes of both sexes. Significant increases occurred in the copper content of whole liver, mitochondria, and cytosol of male rats and in whole liver and cytosol of female rats. Decreases in the copper content of the microsomes of male rats were observed following TCDD treatment; however, TCDD produced no changes in the zinc content of hepatic subcellular fractions of either sex. The magnesium content of female TCDD-treated rats increased in whole liver, mitochondria, and cytosol, while the magnesium content of microsomes was not altered. With respect to the subcellular distribution of iron, copper, zinc, and magnesium, TCDD produces differential effects. The altered distribution of some cations may contribute to the broad range of effects of TCDD.     

Gender‐Related In Vitro Metabolism of Hexaconazole and Its Enantiomers in Rats

In the present study we investigated the enantioselective disappearance of hexaconazole in rat liver microsomes system prepared from both genders. High‐performance liquid chromatography (HPLC) was used for identification and quantification. The degradation of the (+)‐hexaconazole was faster than that of the (?)‐hexaconazole in racemic hexaconazole and single enantiomer incubation in both sexes. The degradation half‐life of the (+)‐hexaconazole or (?)‐hexaconazole was also gender‐related. The metabolism of (+)‐hexaconazole and (?)‐hexaconazole were faster in male rat hepatic microsomes than that in female, suggesting that at least one of the cytochrome P450s (CYP) in the male rat liver microsomes system responsible for hexaconazole metabolism was male‐specific or considerably more active. Kinetic assays showed that the intrinsic clearance in male rat liver microsomes was higher than that in female. All these results strongly suggest that sexual dimorphic metabolism of hexaconazole exists in rats. The inhibition experiments with CYP inhibitors showed that the inhibitory effect of inhibitors was enantioselective and affected by sex. The results suggest that the enantioselective metabolism of hexaconazole was determined by the amount of hepatic cytochrome P450 and the expression of individual isoforms of CYPs. Chirality 25:852–857, 2013. © 2013 Wiley Periodicals, Inc.     

Developmental appearance of proteins identified by two-dimensional gel electrophoresis in mouse gonadal tissue

Gonadal protein patterns of the mouse were studied during fetal development by two-dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post-coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.     

Biochemical differences in Cannabis sativa L. depending on sexual phenotype

Hemp (Cannabis sativa L.) is a species considered as having one of the most complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in leaves of the two sexual phenotypes of hemp were studied. Significant differences in isoperoxidase and isoesterase patterns were found between male and female plants, both in the number and stain intensity of bands. For both esterase and peroxidase, the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious between the two sexes concerning catalase and peroxidase activities, as well as the level of soluble protein. The quantitative analysis of flavones, polyholozides and polyphenols emphasized differences depending not only on sex, but also on tested organ.     

Esterase B1 activity variation within and among insecticide resistant, susceptible and heterozygous strains of Culex quinquefasciatus (Diptera: Culicidae)

Amplification of the esterase B1 gene of Culex quinquefasciatus Say results in high titers of an esterase enzyme that confers resistance to organophosphate insecticides. Esterase activity of individuals was measured in samples from an organophosphate resistant strain (Tem-R), a susceptible strain (S-Lb), and their reciprocal F1 progeny. Within-strain variation, as measured by coefficients of variation, was fairly consistent between sexes within strains and among strains (average, 12%). On average, individuals from the Tem-R strain had about 120 times the esterase activity of individuals from the S-Lab strain. The mean esterase activities of the F1 strains were significantly higher than the average of the Tem-R and S-Lab strain mean esterase activities, suggesting enhanced expression of the amplified esterase B1 genes in F1 individuals. Reciprocal F1 strains did not differ significantly in esterase activity or resistance, indicating that maternal effects do not influence either of these measures in these strains. The levels of esterase activity of the strains are discussed in relation to their resistance.     

Sex-specific constitutive expression of the pre-neoplastic marker glutathione S-transferase, YfYf, in mouse liver.

Hepatic glutathione S-transferase isoenzyme content has been investigated in both sexes of three inbred strains of mice (DBA/2, C3H/He, C57BL6). A polypeptide (Mr 24,800), which is immunologically related to Yf purified from rat lung, was found to be expressed as a major form in all male mouse livers but represented only a minor enzyme form in female mouse liver. Glutathione S-transferases comprising subunits with molecular masses of 25,800 (Ya) or 26,400 (Yb) were present in males and females of the three strains under investigation. Cytosolic isoenzymes from all strains and sexes were purified to apparent homogeneity and no significant inter-strain differences in the properties of the individual forms were observed. In addition, no differences were detected in the microsomal glutathione S-transferase content of the different strains or sexes.     

Sex-related difference in vitamin D3 25-hydroxylase of rat liver microsomes

Cholecalciferol 25-hydroxylase was partially purified by polyethylene glycol fractionation and chromatographies on octylamino-Sepharose and hydroxylapatite columns starting from the liver microsomes of female rats, and compared with P-450cc25 purified from the liver microsomes of male rats (Hayashi, et al. (1986) J. Biochem. 99, 1753-1763). On octylamino-Sepharose 4B column chromatography, most of the activity was recovered in the fraction eluted with 0.08% Emulgen 913 in the case of the male enzyme, whereas the female enzyme was recovered in the fraction eluted with 0.2% Emulgen. Anti-cc25 antibodies against purified male P-450cc25 inhibited the 25-hydroxylation activity of male polyethylene glycol (PEG) fraction and partially purified male enzyme, but did not inhibit the activities of the corresponding female fractions. The antibodies formed a single precipitation line with male P-450cc25, but did not form a precipitation line with partially purified female 25-hydroxylase on immuno-diffusion. These observations indicated that the vitamin D3 25-hydroxylase in female rat liver microsomes is a different entity from that of male rats.     

Tetrahydrofurane - an inhibitor for ethanol-induced liver microsomal cytochrome P450.

Liver microsomes from ethanol-pretreated rats have been compared with microsomes from male and female controls and phenobarbital- and benzpyrene-pretreated rats. The 0-dealkylation activity for 7-ethoxycoumarin was enhanced after all treatments. Metyrapone selectively inhibited the activity after pretreatment with phenobarbital and naphthoflavone blocked the activity after benzpyrene treatment. Ethanol and even more so tetrahydrofurane inhibited specifically the 0-dealkylation in microsomes from ethanol-pretreated rats. Only in these microsomes tetrahydrofurance produced a pronounced ligand-type optical difference spectrum and concomitantly a new low-spin cytochrome P450 species in the EPR-spectrum. According to inhibition experiments, liver microsomes from male and female rats have a different pattern of cytochrome P450 species.     

Schistosoma japonicum: disc electrophoretic protein patterns of the Japanese, Philippine, and Formosan strains

Soluble proteins of the Japanese, Philippine, and Formosan strains of Schistosoma japonicum were separated by disc electrophoresis using polyacrylamide gel columns. Differences between strains and sexes were investigated on the basis of R_f values of the bands, location of prominent peaks, quantitative comparisons of major bands, and overall densitometric profiles. With male extract, 29, 28, and 29, distinct bands were resolved for the Japanese, Philippine, and Formosan strains, respectively. Female extracts gave 31, 26, and 25 distinct bands for the Japanese, Philippine, and Formosan strains, respectively. Both qualitative and quantitative differences were found among the three strains and between sexes. The closest relationship of densitometric profile was between the Japanese and Formosan strains.