Arun P. Kulkarni

OXIDATIVE METABOLISM OF XENOBIOTICS DURING PREGNANCY: SIGNIFICANCE OF MICROSOMAL FLAVIN-CONTAINING MONOOXYGENASE

Pregnancy related changes in oxidative metabolism of model substrates were examined in CD1 mice. As compared to nonpregnant females, a significant decrease in the hepatic microsomal aminopyrine-but not in dimethylaniline-N-demethylase activity was observed in pregnant mice. The rates of microsomal flavin-containing monooxygenase-catalyzed N-oxidation of dimethylaniline remained relatively unchanged during pregnancy in the liver, lung, kidney, and uterus. In contrast to this, N-oxidase activity of placental microsomes was increased nearly 5-fold when measured at day 12 and 18 of gestation.

A rapid, novel high performance liquid chromatography method for the purification of glutathione S-transferase: an application to the human placental enzyme.

A simple High Performance Liquid Chromatography procedure is detailed for the purification of Glutathione S-transferase. The human placental transferase was used to assess its potential. Unlike conventional methods of purification, the procedure is rapid and resolution of the various forms is achieved in less than 20 min. Since recovery is essentially complete, it is possible to isolate different minor forms. Three forms, one major and two minor, were separated. The major form represented about 97% of the total recovered activity and exhibited a specific activity of 254.94 mumoles/min/mg protein with a purification of 1342-fold. Electrophoresis of the major form revealed the presence of a single band, suggesting homogeneity.

HUMAN PLACENTAL LIPID PEROXIDATION. SOME CHARACTERISTICS OF THE NADPH-SUPPORTED MICROSOMAL REACTION

1. The evidence presented in this paper indicates the existence of NADPH-supported lipid peroxidation in human placental microsomes. Thiobarbituric acid assay was used to estimate quantitatively lipid peroxidation. 2. Several biochemical characteristics of the reaction were examined. Maximal lipid peroxidation occurred at pH 7.4 and at a protein concentration of approx. 0.2 mg microsomal protein/ml. The presence of NADPH and chelated iron was required. The reaction was linear up to 5 min and did not exhibit an initial lag phase. 3. Under optimal assay conditions, the rate of lipid peroxidation ranged from 2 to 6 nmol malondialdehyde formed/min/mg protein in different preparations of placental microsomes. 4. Inconclusive results were obtained when assays were performed in the presence of scavengers of reactive oxygen species. 5. Marked inhibition in the malondialdehyde accumulation was observed when phosphate buffer was added to the incubation media. 6. This inhibitory effect appeared to be due to the removal of chelated iron from the system and not due to interference with the electron transport mechanism.

Mutagen activation of 1,2‐dibromo‐3‐chloropropane by cytosolic glutathione s‐transferases and microsomal enzymes

It is not clear whether glutathione (GSH) conjugation to 1,2-dibromo-3-chloropropane (DBCP) results in genotoxic activation. Therefore S9, cytosolic, and microsomal fractions from uninduced rat liver were evaluated for their relative ability to activate DBCP in a modified Ames system. The S9 enzymes, either alone or in combination with exogenous GSH, did not enhance the mutagenicity of DBCP; identical results were obtained with cytosolic enzymes. Significant mutagenic activation of DBCP was produced by either S9 or microsomal fractions in the presence of NADPH. Activation was proportional to cytochrome P-450 concentrations, and was diminished by exogenous GSH. The protection against genotoxicity exerted by GSH did not require cytosolic glutathione S-transferases (GST). Thus, mutagenic activation of DBCP as obtained with S9 fractions is primarily due to biotransformation by microsomal rather than by cytosolic enzymes. Kinetic studies of cytosol-catalyzed conjugation of GSH to DBCP revealed tissue-specific differences in apparent Km and Vmax. Renal and testicular GSTs were associated with 28-46% smaller Vmax values when compared to hepatic GSTs (31.2 +/- 1.9 nmol/min X mg protein). However, renal and testicular GSTs had relatively higher affinities for DBCP. Thus, extrahepatic tissues possess significant capacity to conjugate GSH to DBCP. DBCP-GSH conjugates may undergo enzymatic modification by extrahepatic peptidase and beta-lyase to yield other sulfur-containing moieties that perhaps mediate DBCPs extrahepatic toxicity.

HUMAN PLACENTAL LIPID PEROXIDATION--II. NADPH AND IRON DEPENDENT STIMULATION OF MICROSOMAL LIPID PEROXIDATION BY PARAQUAT

Paraquat, a widely used herbicide, was found to cause a marked stimulation of lipid peroxidation in the human placental microsomes in vitro. Both NADPH and chelated iron were necessary to observe paraquat-stimulated lipid peroxidation. The malondialdehyde accumulation in the incubation medium increased with increase in time, protein and paraquat concentration. The reaction did not exhibit the initial lag phase suggesting that endogenous membrane-bound antioxidants in human placental microsomes are either absent or present in extremely small quantities.

Glutathione S-transferase activity during pregnancy in the mouse: effects of trans-stilbene oxide pretreatment.

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Human placental glutathione S-transferase-mediated metabolism of methyl parathion.

The ability of human placental glutathione S-transferase (GSHTr) to metabolize methyl parathion (MeP) was examined. MeP was found to be a substrate for both partially purified pre-term and highly purified term placental GSHTr. The characterization of the reaction by high performance liquid chromatography revealed the presence of desmethyl parathion (DesMeP) as the sole metabolite. Term placental GSHTr activity towards MeP ranged from 2.22 to 3.53 nmoles DesMeP formed X min-1 X mg-1 while an activity of 0.60 to 1.12 nmoles DesMeP formed X min-1 X mg-1 was observed with the pre-term placental enzyme. The absence of the O-dearylation reaction by pre-term and term placental GSHTr represents a major species- and/or tissue-specific difference.

Enzymatic denitrification of 2-nitropropane in uninduced mouse liver microsomes.

Hepatic microsomes from 5 strains of untreated mice were tested for the ability to enzymatically cleave the nitro group from 2-nitropropane (2NP). All strains showed significant NADPH-dependent nitrite release at pH 7.6 and pH 8.8. Statistical differences in nitrite-releasing activity between strains were found between BALB and PL/J and ATH strains at pH 7.6. At pH 8.8, BIO.M differed from CD-1 and BALB. These results are in contrast to a report of little or no denitrification activity in uninduced rats and suggest that the 2NP microsomal metabolism may be of greater importance than previously thought.

Ethanol potentiation of carbon tetrachloride hepatotoxicity: Possible role for the in vivo inhibition of aldehyde dehydrogenase

A potentiation of CCl4-induced hepatotoxicity was observed in rats pretreated with ethanol 18 hr prior to CCl4 exposure. Hepatic microsomal aldehyde dehydrogenase (ALDH) was significantly inhibited in animals sacrificed 1 hr following the sequential exposure, however, no more so than in those animals receiving CCl4 alone. The animals receiving ethanol alone had ALDH activity similar to vehicle treated controls. Twenty-four hours following a potentiating dose of ethanol and CCl4 an 81 and 57% decline in NAD+-dependent microsomal and mitochondrial ALDH activity was observed, respectively. Similar results were observed for microsomal and mitochondrial NADP+-dependent ALDH activity. The decline in membrane-bound ALDH was greater in potentiated animals than in those receiving CCl4 alone. A relatively smaller decline in cytosolic ALDH activity was observed in CCl4 treated rats with or without ethanol pre-exposure. The data suggest that inhibition of membrane bound ALDH may be one of the major mechanisms of in vivo potentiation of CCl4-induced hepatotoxicity by ethanol.

Hepatic microsomal oxidative metabolism of pesticides and other xenobiotics in pregnant CD1 mice

Abstract Pregnancy-related changes in oxidative metabolism of several xenobiotics including pesticides were examined in the hepatic microsomes of CD 1 mice. The effect of pregnancy on hepatic microsomal cytochrome P -450-catalyzed substrate oxidation was found to be dependent upon the type of reaction examined. Not all substrates undergoing the same reaction showed identical changes during pregnancy. Those enzyme activities which exhibited a decline in specific activity during pregnancy generally exhibited no change in total hepatic capacity. Enzymes posting no change in specific activity throughout gestation generally showed large increases in total hepatic activity. Phorate S -oxidation was catalyzed by both microsomal flavin-containing monooxygenase (MFMO) and cytochrome P -450. Moreover, there was no pregnancy-related change in either MFMO or total enzymatic (MFMO plus cytochrome P -450) phorate S -oxidation.