Untreated Male Rats Research Articles

Effects of Orchidectomy on Nigro‐Striatal Dopaminergic Function: Behavioral and Physiological Evidence

Abstract In the present experiments, we examined the effect of castration upon two indices of nigro-striatal dopaminergic function in the male rat. In Experiment I, differences in spontaneous locomotor behavioral activity between intact and castrated male rats were examined. The total distance traveled, horizontal activity and mean revolutions of castrated male rats were significantly greater than that of intact males. No significant differences between intact and castrated males were obtained for vertical activity. In Experiment II, the spontaneous in vitro dopamine release from the corpus striatum of intact and castrated rats as sampled during the light-phase (1500 h) and dark-phase (2400 h) of the photoperiod was examined. At both time periods, the spontaneous in vitro dopamine release of castrated males was significantly greater than that of intact males. Both intact and castrated males showed statistically significant increases in dopamine release at the 2400 h compared to the 1500 h time period. To examine if testicular hormones were responsible for these castration induced changes in dopamine release, in Experiment III we treated castrated male rats with testosterone propionate. Administration of testosterone propionate (0.1 mg/day x 5 days) significantly reduced in vitro dopamine release compared to untreated or castrated male rats receiving vehicle treatment. These results demonstrate that testicular hormones, most likely testosterone, have a markedly suppressive effect upon the nigro-striatal dopaminergic system as evidenced from changes in spontaneous behavioral activity and in vitro dopamine release.

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats.

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro inhibition of hepatic steroid hydroxylation by tamoxifen, a series of tamoxifen analogues and related compounds

The in vitro inhibition of the cytochrome P-450 (P-450) isozyme specific positional hydroxylation of androst-4-ene-3,17-dione (androstenedione) by the alkylamino containing compounds trans-and cis-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, SKF 525-A and the non-alkylamino containing compounds tamoxifen metabolite E, and tamoxifen analogue U-23469 was assessed in pooled hepatic microsomes isolated from untreated male rats. P-450 IIA 1-mediated androstenedione 7α-hydroxylation appeared refractory to inhibition, with the lowest I 50s being approximately 200 μM ( cis-and trans-tamoxifen, 4-hydroxytamoxifen). (According to the recently recommended nomenclature for cytochromes P-450 (Nebert DW and González FJ, Ann Reu Biochem 56: 945–993, 1987), rat hepatic cytochromes P-450 UT-A, PB-B, PCN-E and UT-F are encoded by genes IIC 11, IIB 1, IIIA 1 2 and IIA 1, respectively. I 50s toward the P-450 IIC 11-, IIB 1-, and IIIA 1 2 -catalysed reactions, androstenedione 16α-, 16β-and 6β-hydroxylations, respectively, were generally in the range 70–190 μM. However, metabolite E exhibited a rather specific and potent capacity to inhibit androstenedione 16α-hydroxylase activity (I 50 = 18 μM). Since a number of alkylamine compounds have been shown to sequester microsomal P-450 as an inactive metabolite intermediate (MI), the tamoxifen analogues were investigated for their in vitro MI complexation capacity. However, spectral binding studies revealed that the incubation of these compounds with NADPH-fortified microsomal fractions did not result in MI complex formation. In binding experiments conducted with oxidised microsomal fractions it was apparent that most of the tamoxifen analogues are type I ligands of quite high affinity for ferric P-450 ( K s range 10–60 μM). It seems unlikely that MI formation is involved in the observed inhibition of androstenedione hydroxylation by tamoxifen and congeners. Instead, and in contrast to the situation observed with SKF 525-A, it would appear that the inhibitory capacity of the tamoxifen analogues is more closely related to type I binding capacity with ferric P-450. A finding of particular interest is that metabolite E, in which the alkylamino side-chain is absent, elicited a type I interaction of high capacity. The maximal absorbance change of the type I interaction of this compound with microsomal P-450 was about three-fold greater than the other compounds. It therefore appears likely that removal of the tamoxifen side-chain results in quite potent inhibition of P-450 IIC 11-mediated monooxygenase activity and an enhanced binding interaction with the cytochrome.

Enantiotopic differentiation during the biotransformation of valproic acid to the hepatotoxic olefin 2-n-propyl-4-pentenoic acid

The enantiomers of 2-[( 3-13C]-n-propyl)pentanoic acid [(R)- and (S)-[13C]VPA] were employed as metabolic probes to investigate stereochemical aspects of the biotransformation of valproic acid (VPA) to 2-n-propyl-4-pentenoic acid (delta 4-VPA), a hepatotoxic metabolite of VPA. When incubated with hepatocytes freshly isolated from untreated male rats, each labeled substrate (initial concentration 1.0 mM) underwent metabolism to [13C]-delta 4-VPA, the formation of which was time-dependent and occurred at a rate of ca. 20 ng/(10(6) cells.4-h incubation). Analysis of this unsaturated metabolite by GC-MS techniques revealed that, following incubation of (R)-[13C]VPA, desaturation had taken place preferentially (by a factor of approximately 4) on the labeled propyl group (i.e., on the R side chain). Parallel incubations with (S)-[13C]VPA supported this conclusion, in that metabolism of this isotopic variant of VPA led to a terminal olefin that also was predominantly (83 +/- 2%) of R configuration (in this case oxidized selectively on the unlabeled side chain). Hence, biotransformation of VPA to delta 4-VPA in rat hepatocytes occurs with marked enantiotopic differentiation, favoring production of the R enantiomer of this chiral metabolite. When rats were pretreated with phenobarbital (80 mg kg-1 day-1 ip for 3 days) prior to isolation of hepatocytes, the overall rate of metabolism of VPA to delta 4-VPA over the 4-h incubation period increased approximately 3-fold, while the degree of product enantioselectivity was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Acinar heterogeneity of fatty acid binding protein expression in the livers of male, female and clofibrate-treated rats.

Liver fatty acid binding protein may play a role in the intracellular transport and compartmentation of long-chain fatty acid metabolism. The distribution of liver fatty acid binding protein in the hepatic acinus was determined by means of immunocytochemistry as well as by measurement of liver fatty acid binding protein in cellular protein selectively released from zone 1 and zone 3 cells by means of anterograde and retrograde liver perfusion with digitonin. In untreated male rats, specific immunocytochemical staining for liver fatty acid binding protein showed a declining portal-to-central hepatocellular gradient in intensity, consistent with the portal-to-central ratio of liver fatty acid binding protein abundance measured in effluents from digitonin-perfused livers of 1.6:1. Female and clofibrate-treated male rats, in both of which hepatic synthesis and abundance of liver fatty acid binding protein are greater than in untreated males, differed as well in the pattern of acinar expression of this protein. In females, periportal concentrations of liver fatty acid binding protein determined from the effluent of livers perfused anterograde with digitonin were similar to male values, whereas liver fatty acid binding protein concentration in pericentral hepatocytes determined from the effluent of retrograde perfused livers was increased, resulting in a marked attenuation of the portal-to-central gradient of this protein; this was also apparent on immunocytochemistry. Clofibrate-treated rats, in contrast, displayed a panacinar increase in liver fatty acid binding protein with maintenance of the portal-to-central ratio observed in untreated males. We conclude that there exists a declining portal-to-central gradient in liver fatty acid binding protein cellular abundance in the hepatic acinus of untreated male rats. Furthermore, the increased synthesis and abundance of liver fatty acid binding protein in female and clofibrate-treated male rats results in two different alterations in the acinar expression of this protein, i.e. a pericentral increase (female) or a panlobular increase (clofibrate). Elucidation of the relationship between the zonation of hepatic fatty acid metabolism and the acinar expression of liver fatty acid binding protein should provide a more detailed understanding of the function of this protein.

Specific profile of liver microsomal cytochrome P-450 in Dolphin and Whales

The cytochrome P-450-linked monooxygenase system in liver microsomes from three species of cetaceans including short-finned pilot whale ( Globicephala macrorhynchus), striped dolphin ( Stenella coeruleoalba) and killer whale ( Orcinus orca) were investigated with reference to the catalysis of the model substrates aldrin, aniline, benzo[a]pyrene, 7-ethoxyresorufin, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), and 2-amino-3,5-dimethylimidazo[4,5-f]-quinoxaline (MeIQ). Cytochrome P-450 contents in cetacean liver microsomes ranged from 0·17 to 0·21 nmol per mg protein. In short-finned pilot whale fetal liver microsomes contained only about 0·04 nmol per mg protein; monooxygenase activities were also lower than those measured in immature or mature individuals. No sex-related differences and developmental changes in cytochrome P-450-linked monooxygenase activities were observed in this species. Interestingly, the cetaceans showed relatively low aldrin epoxidase (ALDE) and aniline hydroxylase (AH) activities, whereas benzo[a]pyrene hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities were observed to be comparable to the activities of liver microsomes from untreated male Sprague-Dawley rats. Polyclonal antibodies raised against a 3-methylcholanthrene (MC) inducible form of rat cytochrome P-450 (P-450c) inhibited AHH and EROD activities and the metabolic activation of MeIQ by liver microsomes from short-finned pilot whales. These results suggest that the cytochrome P-450 isozyme(s) which is (are) orthologous to rat cytochrome P-450c have an important role in some of the drug oxidation reactions in cetacean liver microsomes.

Sex change in cytochrome P-450 phenotype by growth hormone treatment of adult rat hepatocytes maintained in a culture system on matrigel.

Results of studies of hypophysectomized rats suggest that growth hormone serves as a final common mediator through which gonadal steroids and other modifiers of pituitary function alter the expression of gender-specific liver genes such as the sexually dimorphic pair of cytochrome P-450 isozymes, male-specific P-450h and female-specific P-450i. We tested the effects of growth hormone in a system for primary monolayer culture of adult rat hepatocytes on a laminin-rich extracellular matrix (matrigel), which permits sustained expression of both constitutive and inducible liver genes in a chemically defined medium. Cultures of freshly isolated hepatocytes prepared from untreated male rats and samples of the intact donor liver contained readily detectable quantities of immunoreactive P-450h protein (measured on immunoblots of cell microsomes) and P-450h mRNA (measured on Northern blots of cellular RNA). Neither P-450i immunoreactive protein nor P-450i mRNA were present. Addition of physiologic concentrations of human or bovine growth hormone, but not of prolactin, to culture medium lacking insulin or other hormones resulted in prompt induction of P-450i immunoreactive protein and P-450i mRNA. Induction of P-450i mRNA in male hepatocyte cultures was dependent on the concentration of growth hormone, required as little as 24 hr of exposure, and was markedly attenuated in cultures maintained on type I collagen rather than on matrigel. Growth hormone treatment also induced the level of mRNA for insulin-like growth factor I, whereas the amount of mRNA for the male-specific urinary protein alpha 2 mu-globulin was unaffected. Cultures of hepatocytes derived from untreated adult female rats retained high levels of P-450i mRNA but only if the culture medium contained growth hormone. None of the tested treatments with estrogens, androgens, glucocorticoids, or growth hormone induced P-450h mRNA or P-450h immunoreactive protein in cultures of female hepatocytes. We conclude that the somatogenic effects of growth hormone acting alone and directly on the hepatocyte in culture are sufficient to "feminize" the cytochrome P-450 phenotype. The present culture system offers a way to explore the molecular basis for hormonal control of liver gene expression.

Four species of cDNAs for cytochrome P450 isozymes immunorelated to P450C-M/F encode for members of P450IID subfamily, increasing the number of members within the subfamily

A chicken antibody mono-specific to cytochrome P450C-M/F, which exists in untreated male and female rat liver and catalyses the 2- and 16 alpha-hydroxylation of estrogens (1), was used to screen a cDNA library of male Sprague-Dawley rat liver. Four cDNA clones which encoded P450 isozymes, CMF1a, CMF1b, CMF2 and CMF3, were isolated. CMF1a and CMF2 deduced consisted of 504 and 500 amino acid residues, respectively, while C-terminal 487 and 324 residues for CMF1b and CMF3, respectively, were deduced from the 5'-truncated cDNAs. The isozymes were more than 72% similar in amino acid sequences to each other and to rat P450db1, P450db2 (2), and to a mouse male specific C-P45016 alpha (3), suggesting that they belonged to a new P450 subfamily, P450IID. CMF1a and db1, and CMF2 and db2, respectively, were 99.2% and 99.0% similar in amino acid sequences, suggesting that they were virtually identical. CMF1a and CMF1b were different but 96.1% similar, and CMF3 was between 76% and 78% similar to other members of the rat P450IID family.

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal administrations of a vasopressin analog (DDAVP) and hypertonic saline enhance learning behavior in rats

Groups of newborn Wistar rats received daily 1-desamino-8-D-arginine-vasopressin (DDAVP), oxytocin (OXT), hypertonic saline or normal saline for 14 days from day 1 to day 14 of life. One or three months later they were trained in a maze for brightness discrimination (BD). A group of untreated adult male rats received posttrial DDAVP or normal saline for brightness discrimination. Subsequently all the retentions of BD were tested after one month. We found that the neonatal treatments with both DDAVP and hypertonic saline facilitated acquisition and subsequent maintenance of brightness discrimination in immature and mature rats, and also that posttreatment with DDAVP enhanced retention of BD in adult rats. Oxytocin and normal saline had no effect on these parameters. The results are interpreted as showing that endogenous AVP and its synthetic analog enhance the development and adult function of central neural substrates involved in learning behaviors.

Effect of thyroid status and paraventricular area lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood.

TRH is a potent stimulator of pituitary TSH release, but its function in the physiological regulation of thyroid activity is still controversial. The purpose of the present study was to investigate TRH and catecholamine secretion into hypophysial portal blood of hypothyroid and hyperthyroid rats, and in rats bearing paraventricular area lesions. Male rats were made hypothyroid with methimazole (0.05% in drinking water) or hyperthyroid by daily injections with T4 (10 micrograms/100 g BW). Untreated male rats served as euthyroid controls. On day 8 of treatment they were anesthetized to collect peripheral and hypophysial stalk blood. In euthyroid, hypothyroid and hyperthyroid rats plasma T3 was 1.21 +/- 0.04, 0.60 +/- 0.04, and 7.54 +/- 0.33 nmol/liter, plasma T4 50 +/- 3, 16 +/- 2, and 609 +/- 74 nmol/liter, and plasma TSH 1.58 +/- 0.29, 8.79 +/- 1.30, and 0.44 +/- 0.03 ng RP-2/ml, respectively. Compared with controls, hyperthyroidism reduced hypothalamic TRH release (0.8 +/- 0.1 vs. 1.5 +/- 0.2 ng/h) but was without effect on catecholamine release. Hypothyroidism did not alter TRH release, but the release of dopamine increased 2-fold and that of noradrenaline decreased by 20%. Hypothalamic TRH content was not affected by the thyroid status, but dopamine content in the hypothalamus decreased by 25% in hypothyroid rats. Twelve days after placement of bilateral electrolytic lesions in the paraventricular area plasma thyroid hormones and TSH levels were lower than in control rats (T3: 0.82 +/- 0.05 vs. 1.49 +/- 0.07 nmol/liter; T4: 32 +/- 4 vs. 66 +/- 3 nmol/liter; TSH: 1.08 +/- 0.17 vs. 3.31 +/- 0.82 ng/ml). TRH release in stalk blood in rats with lesions was 15% of that of controls, whereas dopamine and adrenaline release had increased by 50% and 40%, respectively. These results suggest that part of the feedback action of thyroid hormones is exerted at the level of the hypothalamus. Furthermore, TRH seems an important drive for normal TSH secretion by the anterior pituitary gland, and thyroid hormones seem to affect the hypothalamic release of catecholamines.

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: Formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the ω, ω-1, and ω-2 hydroxylation of prostaglandins [ K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ]. The current study examined the regioselectivity of hydroxylation of PGE 1 and PGE 2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE 1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH- P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b 5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE 1, RLM 5 catalyzed solely the ω-1 hydroxylation and 5a catalyzed primarily the ω-1 and little ω and ω-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE 1 and PGE 2 at the ω-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is ω-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE 1 hydroxylation demonstrated that the affinity of f4 for PGE 1 is approximately 100-fold higher than that of RLM 5; the K m values for f4, monitoring 19- and 17-hydroxylation of PGE 1, were about 10 μ m. Surprisingly, cytochrome b 5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE 2 by RLM 5 was at the ω, ω-1, and ω-2 sites, demonstrating a lesser regioselectivity than with PGE 1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b 5 requirement.

Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen.

Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-S-ylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH-APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-450UT-A) catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P-450UT-A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P-450UT-F and the female specific isozyme P-450UT-I, produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by beta-naphthoflavone, cytochrome P-450 beta NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-450PB-B) a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450PB-B and P-450 beta NF-B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial mechanical and myosin isoenzyme alterations in streptozotocin-diabetic rats.

Fifteen week old male Wistar rats (n = 7) were made diabetic by intravenous injection of streptozotocin (50 mg/kg). Age-matched, untreated male Wistar rats (n = 9) served as controls. Hearts were removed after 5-6 weeks of diabetes, and the isometric developed tension (T) of isolated left ventricular papillary muscles and its first derivative (dT/dt) were measured at a frequency of 0.2 Hz. During testing, the muscles were perfused with Tyrode's solution (Ca2+ concentration was half of normal Tyrode's solution, pH 7.4, 32 degrees C, bubbled with 95% O2 and 5% CO2). In addition, the left ventricular isoenzyme pattern, which is related to myocardial energetics, was determined by pyrophosphate gel electrophoresis. There was no significant difference in isometric developed tension between diabetic and control rats (DM: 2.90 +/- 0.89 vs controls: 2.87 +/- 0.85 g/mm2, mean +/- SD), but in diabetic rats, dT/dtmax decreased significantly as compared with controls (DM: 23.5 +/- 4.2 vs controls: 31.9 +/- 7.9 g/mm2.s, p less than 0.05). Myocardial mechanical responses to isoproterenol (10(-7)M) and dibutyryl cyclic AMP (10(-5)M) also decreased in diabetic rats. The left ventricular myosin isoenzyme pattern shifted toward VM-3 in diabetic rats (VM-3: DM: 74.9 +/- 10.7 vs controls: 9.5 +/- 4.1%, p less than 0.001). These results indicate that diabetes influences myocardial contractility and changes cardiac energetics. Post-receptor processes may play a role in myocardial mechanical responses to catecholamines in streptozotocin-diabetic rats.

Increases in cytochrome P-450 mediated 17 beta-estradiol 2-hydroxylase activity in rat liver microsomes after both acute administration and subchronic administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a two-stage hepatocarcinogenesis model.

Administration of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 micrograms/kg) increased estradiol 2-hydroxylase (E2OHase) activity approximately 2-fold in liver microsomes of female rats but had no effect on E2OHase activity in hepatic microsomes of male rats. In contrast, TCDD increased P-450d (an enzyme which has a high turnover number for E2OHase in a reconstituted enzyme system) 10- to 20-fold in livers of both male and female rats. The discrepancy between the increases in P-450d and E2OHase activity in liver microsomes of TCDD-induced rats was abolished by the addition of exogenous purified P-450 reductase to the microsomal assays for E2OHase, suggesting that reductase was limiting in the in vitro assays. When E2OHase activity was assayed in the presence of exogenous reductase, TCDD increased E2OHase 2-fold and 4-fold respectively in liver microsomes of male and female rates. Antibody to P-450d completely inhibited the increase in E2OHase activity in liver microsomes of TCDD-treated male and female rats, but had little effect on E2OHase activity in liver microsomes of untreated male or female rats. These data indicate that P-450d is responsible for the increase in E2OHase activity in TCDD-treated rats, but other P-450 isozymes are responsible for constitutive E2OHase activity. Biweekly administration of 1.4 micrograms/kg of TCDD for 30 weeks as a potential promoter of hepatocarcinogenesis increased the volume of the liver occupied by gamma-glutamyl transpeptidase (GGT)-positive foci in livers of female rats given a necrogenic dose of diethylnitrosamine (DEN) (200 mg/kg) as the initiator. Biweekly doses of 0.14-1.4 micrograms/kg TCDD in this model also increased P-450d (7-fold) and E2OHase activity maximally (4-fold) in DEN-initiated rats. Moreover, initiation with DEN substantially enhanced the effects of the low dose of TCDD on both hepatic microsomal P-450d and E2OHase activity.

Immunohistochemical localization of cytochrome P-450 in the rat brain

Immunohistochemical localization of cytochrome P-450 in the brains of normal untreated male rats has been demonstrated with polyclonal antibodies raised against hepatic P-450C. These antibodies recognize P-450C and D, two closely related forms of P-450. We have identified P-450C,D immunoreactivity (P-450C,D-i) within both neurons and glial cells. Neuronal cell bodies showing P-450C,D-i were present in several forebrain areas, including the olfactory bulb, the caudate nucleus, the cingulate, the frontal and the entorhinal cortex, the hippocampus and the thalamus. In the brainstem, P-450C,D-i neurons were detected in the substantia nigra, the nucleus locus coeruleus, raphe nucleus, reticular formation and the trigeminal nerve nucleus and the dorsal motor nucleus of the vagus. Glial cells containing P-450C,D-i were situated within myelinated fiber tracts in the forebrain (e.g. the corpus callosum, the internal capsule) and the brainstem (e.g. the superior cerebellar peduncle, the medial longitudinal fasciculus). Some, but not all, of the P-450C,D-i glial cells also stained with an antibody against glial fibrillary acidic protein. The present findings represent the first immunohistochemical demonstration of cytochrome P-450 in the brain and suggest that cytochrome P-450C,D is constitutive in neurons and glial cells of most brain regions throughout the rat neuraxis.

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH 2-terminal sequence and catalytic activity of them were determined. The NH 2-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH 2-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetmaine, 450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 ad P450 UT-2 were active in the 2α- and 16α-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6β-, 7α- and 15α-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.

Isozyme specificity of testosterone 7α-hydroxylation in rat hepatic microsomes: Is cytochrome P-450a the sole catalyst?

In the present study we show that monospecific antibody against cytochrome P-450a completely inhibits testosterone 7α-hydroxylation in hepatic microsomes of untreated male or female rats or rats of either sex treated with dexamethasone. These data are in contrast with those of K. Nagata et al. (1987, J. Biol. Chem. 262, 2787–2793) who recently reported that an antibody prepared against cytochrome P-450a completely inhibited testosterone 7α-hydroxylase activity in microsomes from untreated or 3-methylcholanthrene-treated rats but only inhibited 50% of the activity in microsomes from dexamethasone-treated rats. They proposed that dexamethasone treatment of rats induced another testosterone 7α-hydroxylase in rat liver. The discrepancy in the two sets of data was due, at least in part, to the use of a chromatography system by Nagata et al. that is incapable of resolving a number of testosterone metabolites. Dexamethasone treatment of rats leads to a marked increase in the production of several testosterone metabolites, including 15β-hydroxytestosterone which is cochromatographic with 7α-hydroxytestosterone in their chromatography system. Our results indicate that cytochrome P-450a accounts for all of the testosterone 7α-hydroxylase activity in microsomes from dexamethasone-treated rats, and that testosterone 7α-hydroxylation continues to be a useful marker for monitoring cytochrome P-450a in rat hepatic microsomes.

Genotoxic and mutagenic activation of aflatoxin B 1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B 1 (AFB 1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB 1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB 1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB 1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/ S. typhimurium TA98 system by activated metabolites of AFB 1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB 1 in rat liver microsomes.

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes.

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.