Adult Rat Liver Microsomes Research Articles

Age dependent in vitro metabolism of bifenthrin in rat and human hepatic microsomes

Bifenthrin, a pyrethroid insecticide, undergoes oxidative metabolism leading to the formation of 4′-hydroxy-bifenthrin (4′-OH-BIF) and hydrolysis leading to the formation of TFP acid in rat and human hepatic microsomes. In this study, age-dependent metabolism of bifenthrin in rats and humans were determined via the rates of formation of 4′-OH-BIF and TFP acid following incubation of bifenthrin in juvenile and adult rat (PND 15 and PND 90) and human (<5years and >18years) liver microsomes. Furthermore, in vitro hepatic intrinsic clearance (CLint) of bifenthrin was determined by substrate consumption method in a separate experiment. The mean Vmax(±SD) for the formation of 4′-OH-BIF in juvenile rat hepatic microsomes was 25.0±1.5pmol/min/mg which was significantly lower (p<0.01) compared to that of adult rats (86.0±17.7pmol/min/mg). However, the mean Km values for juvenile (19.9±6.6μM) and adult (23.9±0.4μM) rat liver microsomes were similar. On the other hand, in juvenile human hepatic microsomes, Vmax for the formation of 4′-OH-BIF (73.9±7.5pmol/min/mg) was significantly higher (p<0.05) than that of adults (21.6±0.6pmol/min/mg) albeit similar Km values (10.5±2.8μM and 8.9±0.6μM) between the two age groups. The trends in the formation kinetics of TFP acid were similar to those of 4′-OH-BIF between the species and age groups, although the differences between juveniles and adults were less pronounced. The data also show that metabolism of bifenthrin occurs primarily via oxidative pathway with relatively lesser contribution (~30%) from hydrolytic pathway in both rat and human liver microsomes. The CLint values for bifenthrin, determined by monitoring the consumption of substrate, in juvenile and adult rat liver microsomes fortified with NADPH were 42.0±7.2 and 166.7±20.5μl/min/mg, respectively, and the corresponding values for human liver microsomes were 76.0±4.0 and 21.3±1.2μl/min/mg, respectively. The data suggest a major species difference in the age dependent metabolism of bifenthrin. In human liver microsomes, bifenthrin is metabolized at a much higher rate in juveniles than in adults, while the opposite appears to be true in rat liver microsomes.

Sex‐specific effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic/steroid‐metabolizing enzymes and antioxidant status

Liver has evolved complex enzymatic mechanisms to detoxify a wide array of xenobiotic substances, ranging from dietary components to environmental toxins to pharmaceuticals. Activities of many steroid-metabolizing enzymes in adult rat liver microsomes are sexually differentiated. Toxic effects of lead and cadmium on hepatic tissue have been well established in our earlier studies. We thus monitored the effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic- and steroid-metabolizing enzyme activities in both male and female F1 generation postnatal day (PND) 56 rats. Adult pregnant female rats were treated subcutaneously [0.05 mg/(kg body wt. day)] with sodium acetate (control group), lead acetate, and cadmium acetate separately and in combination throughout the gestational and lactational period. Hepatic phase I xenobiotic-metabolizing enzymes (NADPH- and NADH-cytochrome c reductase) activities significantly decreased significantly in all the metal-treated groups in both PND 56 male and female rats as compared with the control group. Hepatic phase II enzymes (uridine diphosphate-glucuronosyl transferase, gamma-glutamyl transpeptidase, glutathione-S-transferase, 17-beta-hydroxysteroid oxidoreductase) were also highly susceptible to all the metal-treated groups. The observed alterations in the oxidative stress and biochemical parameters in the liver of F1 generation male and female rats resulted from an independent effect of lead and/or cadmium and also from their interaction. Results suggest that early developmental exposure to lead and cadmium both alone and in combination can suppress the hepatic xenobiotic-metabolizing enzyme activities in the liver of F1 generation male and female rats in a sex-dependent manner.

Developmental increases in rat hepatic microsomal UDP-glucuronosyltransferase activities toward xenoestrogens and decreases during pregnancy.

Xenoestrogens, such as bisphenol A and diethylstilbestrol, are glucuronidated by an isoform of UDP-glucuronosyltransferase named UGT2B1 in the livers of adult male rats. In this study, we found that nonylphenol and octylphenol are also conjugated with glucuronic acid by adult rat liver microsomes. Although UDP-glucuronosyltransferase activities toward these xenoestrogens were not detected in the fetal rat liver, a linear increase in enzymatic activities during neonatal development was observed. At 3 weeks after birth, the activities had reached the same level as that of adult rats. The protein and mRNA contents of UGT2B1 also were not detected in the fetal rat liver, but a developmental increase in newborn rat liver was detected by Western and Northern blotting analysis. Additionally, rat hepatic microsomal UDP-glucuronosyltransferase activities toward these xenoestrogens were reduced by about half during pregnancy of mother rats. The results suggest that the reproductive organs of fetal and early-stage neonatal rats, which are sensitive to sex hormones, face a high risk of exposure to free active xenoestrogens.

Self-augmentation Effect of Male-specific Products on Sexually Differentiated Progesterone Metabolism in Adult Male Rat Liver Microsomes

It is well known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific or -predominant manner by adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16alpha-hydroxyl and 6beta-hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5alpha-reduced products by female-predominant 5alpha-reductase in the female. These sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control. In the present study, we show that unlabeled 16alpha-hydroxyprogesterone and 6beta-hydroxyprogesterone inhibited the 5alpha-reductive [(3)H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3,20-dione not only stimulated the 5alpha-reductive metabolism producing themselves but also inhibited the male-specific oxidative metabolism. This finding compels us to propose a novel hypothesis that adult male rat liver microsomes may possess a self-augmentation system regulated by the male-specific products on sexually differentiated steroid metabolism, besides regulation by gene expressions of the related enzymes.

Conformational Change of the Catalytic Subunit of Glucose-6-phosphatase in Rat Liver during the Fetal-to-Neonatal Transition

The glucose-6-phosphatase system was investigated in fetal rat liver microsomal vesicles. Several observations indicate that the orientation of the catalytic subunit is different in the fetal liver in comparison with the adult form: (i) the phosphohydrolase activity was not latent using glucose-6-phosphate as substrate, and in the case of other phosphoesters it was less latent; (ii) the intravesicular accumulation of glucose upon glucose-6-phosphate hydrolysis was lower; (iii) the size of the intravesicular glucose-6-phosphate pool was independent of the glucose-6-phosphatase activities; (iv) antibody against the loop containing the proposed catalytic site of the enzyme inhibited the phosphohydrolase activity in fetal but not in adult rat liver microsomes. Glucose-6-phosphate, phosphate, and glucose uptake could be detected by both light scattering and/or rapid filtration method in fetal liver microsomes; however, the intravesicular glucose-6-phosphate and glucose accessible spaces were proportionally smaller than in adult rat liver microsomes. These data demonstrate that the components of the glucose-6-phosphatase system are already present, although to a lower extent, in fetal liver, but they are functionally uncoupled by the extravesicular orientation of the catalytic subunit.

A Novel Reverse Phase HPLC Method to Separate and Quantify Androstenedione and Its Stereospecific Hydroxyaromatic Derivatives

Abstract Reverse - phase High Pressure Liquid Chromatography with a gradient elution on a LiChrocart 250-4 LiChrospher 100 RP-18 column has been used to separate and quantify 7 α-hydroxyandrostenedione (7α-OHA), 6β-hydroxyandrostenedione (6β- OHA), 16α-hydroxyandrostenedione (16α - OHA), 2 β - hydroxyandrostenedione (2 β - OHA), testosterone (T) and androstenedione (A). These steroids are the major products of androstenedione hydroxylation by adult rat liver microsomes. Separation was achieved in 30 minutes by using a linear gradient of acetonitrile (CH3CN) and water in increasing amounts from 30% to 60% of the first solvent (CH3CN). This new method compares very favourable with other methods already reported for studying microsomal hydroxylations of androstenedione in different positions of the steroidal skeleton.

Fetal and adult human liver differ markedly in the fluidity and lipid composition of their microsomal membranes.

The fluidity and lipid composition of the human hepatic microsomal membrane were studied in 11 livers from 16- to 21-week-old fetuses and in 5 adult livers, and compared with those of fetal and adult rat liver microsomes. Membrane fluidity was analyzed by measurement of fluorescence polarization using the fluorophore 1,6-diphenyl-1,3,5-hexatriene. The lipid apparent microviscosity (eta) of human fetal liver microsomes was 2.17 +/- 0.13 poise, as compared with 1.08 +/- 0.08 poise in adult liver (p less than 0.001). Similar differences in fluidity were found between fetal and adult rat liver microsomes. The more "fluid" adult microsomes had higher phospholipid/cholesterol and phosphatidylcholine/sphingomyelin molar ratios than those of the more "rigid" fetal microsomes. The degree of unsaturation of the adult microsomal lipids was much higher than that of the fetal lipids. The ratios of unsaturated/saturated fatty acids in microsomal lipids highly correlated with the eta values obtained for the combined group of fetal and adult human livers, suggesting that the developmental increase in degree of unsaturation of the microsomal lipids is a major determinant of the increased fluidity of adult as compared with fetal liver microsomes. These differences in fluidity and lipid composition between fetal and adult human liver microsomes may be a critical factor in the regulation of hepatic microsomal drug and carcinogen metabolizing enzyme activity, and could so determine the extent of toxicity and teratogenicity of drugs and/or their metabolites in the developing human fetus.

An effect of estradiol and testosterone on the calcium pump activity and phospholipid fatty acid composition of rat liver microsomes.

Castration of the male rat resulted in a 28% reduction of the specific activity of liver microsomal calcium uptake, three weeks after castration. Treatment of the castrated animals with testosterone during this period returned calcium uptake to control levels. Treatment with estradiol resulted in a reduction of calcium uptake to a level less than 25% of that seen in the normal male. Although testosterone treatment had only a small effect on the fatty acid composition of liver microsomal phospholipids in the castrated male, there were significant changes of linoleic acid (18:2) in phosphatidylcholines and of palmitic acid (16:0) in phosphatidylethanolamines, when compared to the untreated castrated male rat. Administration of estradiol to the castrated male rat resulted in a marked decrease of palmitic acid (16.0) and linoleic acid (18.2) in all three phospholipid fractions studied. Stearic acid (18.0) was significantly increased in the phosphatidylcholines and phosphatidylethanolamines by estradiol treatment. The phospholipid and calcium uptake changes seen after treatment of the castrated rat with testosterone or estradiol are consistent with the sex-related differences observed in the intact, adult rat liver microsomes.

MULTIPLICITY OF CYTOCHROMES P-450 DURING DEVELOPMENT

Drug oxidations are catalyzed by the cytochromes P-450 monooxygenase complex. Livers of induced adult animals possess multiple forms of the hemoprotein component (P-450s) with distinct substrate and product specificities (Mol. Pharmacol., 11:874, 1975). Prompted by (1) a difference in neonatal pharmacokinetic profiles between caffeine and theophylline and most other ‘oxidized’ drugs (phenytoin, etc.) and (2) a reported difference in the substrate specificity of human fetal and adult hepatic monooxygenase activity, we are assessing individual P-450s in untreated and developing organisms. Hepatic microsomes from adult or 72-hr guinea pigs are treated for 3 hrs at 23° in a buffer containing glycerol, cholate, and Emulgen 911, a nonionic detergent. The solubilized P-450s, measured by CO binding, have mol. wts. of ca. 50,000 upon gel filtration. These P-450s can be separated from other hemoproteins and resolved into 4 peaks by DE52 chromatography with ca. 50% recovery. Each peak, when isoelectrically focused in poly-acrylamide gels, can be further resolved into multiple protein bands, 2-4 of which stain for heme. Mixing experiments support this multiplicity. Similar results are obtained with adult rat liver microsomes. Guinea pigs, which exhibit substantial increases in hepatic monooxygenase activity within 72 hrs of birth also exhibit at this time multiple forms of P-450s as assessed by the above methods. Comparisons of gels and elution patterns of adult and 72 hr solubilized P-450s suggest that the relative proportions of multiple forms vary during development.