Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Neonatal phenobarbital administration results in increased cytochrome P450-dependent monooxygenase activity in adult male and female rats

The effects of neonatal exposure to phenobarbital during the first five days after birth on the enzymatic activity of the adult male and female rat liver P₄₅₀-dependent monooxygenase system were investigated. Although liver weight per 100 grams of body weight and total hepatic microsomal protein content were not altered in adult rats treated neonatally with phenobarbital, both sexes did show significant increases in cytochrome P₄₅₀ content, cytochrome P₄₅₀ reductase activity, cytochrome c reductase activity, ethoxycoumarin-O-deethylase activity and in the activity of a specific glucuronyl-transferase. Several of these activities were increased to a larger extent in the females, suggesting that females may be more sensitive to this phenomenon.     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline     

Modulation of hepatic glucose-6-phosphate dehydrogenase activity in male and female rats by estrogen

The effect of estradiol-17 beta on the activity of glucose-6-phosphate dehydrogenase was studied in both male and female rats to further characterize the sex differences in the activity of this enzyme. Four groups of intact and castrated rats were implanted subcutaneously with graded doses (2.4, 4.8 and 7.2 micrograms/day) of pelleted estradiol in a physiologically relevant experimental system. After fourteen days the rats were sacrificed and their livers were assayed for G6PD activities. The result indicated that: (i) the enzyme activity was 3-fold higher in normal adult female than in male rats, (ii) low doses of E2 (2.4, 4.8 and 7.2 micrograms/day) increased the activity of G6PD 6-fold in castrated males and over 2-fold in female castrates as well as intact rats (iii) E2 stimulation of G6PD activity appears to be more effective in castrated males than in female rats (IV) sex difference in the activity of G6PD disappeared after treatment with E2 in castrated rats. It is concluded that the activity of G6PD in rats is markedly enhanced by low doses of E2, which appears to be largely responsible for the sex differences in the activity of this enzyme in rats.     

The effects of nonadecafluoro-n-decanoic acid on serum retinol and hepatic retinyl palmitate hydrolase activity in male Sprague-Dawley rats

The effects of nonadecafluoro-n-decanoic acid (NDFDA) on serum retinol levels and hepatic retinyl palmitate hydrolase (RPH) activity were investigated in male Sprague-Dawley rats given a single intraperitoneal (IP) dose of 0, 50, or 100 mg/kg NDFDA and sacrificed at two, eight, or 11 days. Treated animals exhibited depressed serum retinol levels, lymphoid involution, and failure to gain weight in proportion to the dose. Hepatic RPH activities were depressed in both treatment groups at all time points and correlated with serum retinol levels. Hepatic retinol levels were also depressed by Day 11. Extraction of hepatic homogenates with acetone removed NDFDA and increased RPH activities twofold and threefold for the low- and high-dose groups, respectively. Analysis of partially purified RPH showed both NDFDA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to be noncompetitive inhibitors: KI = 450 and 750 microM, respectively. We conclude that NDFDA causes a decrease in the mobilization of vitamin A from the liver by noncompetitive inhibition of RPH.     

Thyroid C cells in male and female rats with chronic renal failure

The kidneys are responsible for iodine and of thyroid hormone biodegradation. The aim of this study was the histomorphological and immunohistochemical evaluation of the influence of sex on parafollicular thyroid C cells in rats with chronic renal failure. The experiment included 40 Wistar rats after subtotal nephrectomy, after sham operation, and without any surgical procedure. Two weeks after nephrectomy, fragments of thyroids were collected from the examined animals. Paraffin sections were stained with H+E and by silver impregnation. Calcitonin (CT), synaptophysin (SPh), somatostatin (ST), and neuron-specific enolase (NSE) were detected immunohistochemically in C cells. In rats with experimental uremia, immunostaining for the examined substances increased significantly in comparison to the controls. We also observed higher number of C cells with a stronger reaction in the group of males, compared to the female rats.     

Thyroid hormone regulation by stress and behavioral differences in adult male rats

Thyroid hormones are essential regulators of growth, development and normal bodily function and their release is coordinated by the hypothalamic-pituitary-thyroid (HPT) axis. While the HPT axis has been established as an acutely stress-responsive neuroendocrine system, relatively little is known about the mechanisms of its stress regulation. The present study examined acute stress-induced changes in peripheral hormone levels [triiodothyronine (T3); thyroxine (T4), thyroid-stimulating hormone (TSH), reverse triiodothyronine (rT3)] and central mRNA levels of regulators of the HPT axis [thyrotropin-releasing hormone (TRH), somatostatin (SST), type II deiodinase (D2)] in response to an inescapable tail-shock, a rodent model of stress. Additionally, we examined whether individual differences in spontaneous exploratory behavior in an open field test predicted basal levels of TH or differential susceptibility to the effects of stress. The stress condition was associated with decreases in peripheral T3, T4 and TSH, but not rT3, when compared with controls. No changes were observed in TRH or SST mRNA levels, but there was a trend suggesting stress-related increases in D2 mRNA. We also found that an animal's exploratory behavior in an unfamiliar open field arena was positively related to peripheral thyroid hormone levels and predicted the magnitude of stress-induced changes.In conclusion, we found suggestive evidence for stress-induced decrease in central drive HPT axis, but the central mechanisms of its stress regulation remain to be elucidated. Additionally, we found that individual differences in animals' exploratory behavior were correlated with peripheral TH levels.     

Characterization of the effects of known hepatic monooxygenase inducers on male and female rat and mouse kidneys

Few studies have been designed to quantify the response of the mammalian kidney to agents known to induce monooxygenase activity of renal monooxygenase response to three agents representing different classes of inducers: 2,4,2',4'-tetrachlorobiphenyl (2,4,2',4',-TCB), representative of the barbiturate class, beta-naphthoflavone (BNF), representative of the polycyclic aromatic hydrocarbon class and isosafrole (ISO) as a novel class of inducing agent. Studies were carried out using adult rats and mice of both sexes. Treatment with BNF and ISO stimulated ethoxycoumarin and ethoxyresorufin deethylase activities in renal microsomes from male and female rats and mice, whereas treatment with 2,4,2',4',-TCB had no effect on either enzyme in rats of either sex. NADPH-cytochrome-c-reductase activity was unaffected by any treatment. In rat renal microsomes, cytochromes P-450 and b5 were increased by treatment with BNF and ISO but were not altered by 2,4,2',4'-TCB. Sodium dodecyl sulphate-polyacrylamide gel treated with BNF showed the appearance of a protein band in the 50-60000 dalton range which is similar to that observed in liver microsomes following BNF treatment. These studies confirm and extend previous observations that rat kidney is refractory to induction by inducers of the phenobarbital class, but responds to ISO and the polycyclic aromatic class of inducers. In addition, the studies have demonstrated the presence of a protein in renal microsomes after pretreatment of rats with BNF that was not apparent in microsomes from control rat kidneys.     

Induction of a phosphate appetite in adult male and female rats

We have reported that dietary inorganic phosphate (Pi) deprivation induces a Pi-seeking behavior in juvenile male rats. The purpose of the present study was to determine whether the Pi appetite is present in adult animals, and if so, whether it is altered during times of increased demand for Pi, such as pregnancy and lactation. Both male and female animals fed a low-phosphate diet (LPD) ingested significantly greater amounts of PiH(2)O daily than their normal phosphate diet (NPD) controls, and per 100 g of body weight (BW), the female animals fed LPD tended to ingest greater amounts of PiH(2)O than male rats fed LPD. Pregnant and lactating rats fed LPD ingested significantly more PiH(2)O than those fed NPD, however, neither group displayed a Pi appetite different than virgin females. However, lactation further reduced Pi levels in plasma and cerebral spinal fluid compared with control values. Despite the additional Pi from the PiH(2)O in the mothers fed LPD, pup birth weight was significantly lower than in NPD litters, and this was exacerbated 9 days after birth. This attenuated BW gain was associated with lower plasma Pi levels in the pups. In conclusion, a mild but consistent Pi-seeking behavior is induced in adult male and female rats after only 2 days of dietary Pi restriction. On a relative basis, the amount of PiH(2)O ingested is greater in female than in male animals, but does not increase further during pregnancy and lactation.     

The heme oxygenase system and its functions in the brain.

The heme oxygenase (HO) system was identified in the early 1970s as a distinct microsomal enzyme system that catalyzes formation of bile pigments (Maines and Kappas, 1974). Up to the early 1990s the system was considered only as a "molecular wrecking ball" (Lane, 1998) for degradation of the heme molecule and production of toxic waste products, CO and bile pigments. For those years, the HO system remained relatively unknown to the research community. In a rather short span of the past 10 years following the discovery of high levels of a second form of the enzyme, HO-2, in the brain, suggesting that "heme oxygenase in the brain has functions aside from heme degradation" (Sun et al., 1990); concomitant with finding that another toxic gas, NO, is a signal molecule for generation of cGMP (Ignarro et al., 1982), the system was propelled into main stream research. This propulsion was fueled by the realization of the multiple and diverse functions of heme degradation products. Heme oxygenase has now found relevance in all kinds of human pathophysiology ranging from stroke, cancer, multiple sclerosis, and malaria to transplantation and immune response. As it turns out, its potential benefits are mesmerizing investigators in diverse fields (Lane, 1998). The most recent findings with HO-2 being a hemoprotein and potentially an intracellular "sink" for NO (McCoubrey et al., 1997a; Ding et al., 1999), together with the discovery of the third form of the enzyme, HO-3 (McCoubrey et al., 1997b), are likely to insure the widespread interest in the enzyme system in the coming years. The present review is intended to highlight molecular properties of HO isozymes and their likely functions in the brain. Extended reviews of the system are found in Maines (1992, 1997).     

Prolactin-releasing effect of tryptolines in the developing and adult male and female rats

The developmental prolactin-releasing effect of Tryptoline (T), Methoxytryptoline (MT) and Hydroxytryptoline (OHT) was examined comparatively in male and female rats. A single injection of T 15 mg/Kg increased serum prolactin in both sexes; the increase was significant from day 20 onwards. OHT evoked a sharp rise in 12 day-old rats and the releasing effect increased with age, both in males and females. No significant sex differences were observed in T or OHT treated rats. MT caused an increment in prolactin secretion in male rats and this action increased with age. The releasing effect of MT was not significant in females, even at 38 postnatal days. In adult animals, the tryptolines (15 mg/Kg) were able to increase serum prolactin in males and in females in diestrous; a dose of 5 mg/Kg of T was only effective in adult male rats. The prolactin-releasing effect was drastically reduced by orchidectomy and by ovariectomy. LH, FSH and TSH were not modified by any treatment. The present results show for the first time the ontogeny of the prolactin-releasing effect of tryptolines in male and female rats and that this effect depends on the presence of gonadal secretions in adults.     

Intrauterine contiguity influences regulatory activity in adult female and male mice

Female mice located in utero between two female fetuses exhibited higher levels of locomotor activity in adulthood than did females located between two male fetuses. Male mice, which were less active than females, also were influenced by intrauterine contiguity. Males located in utero between two female fetuses were more active than males which resided between two male fetuses. These results indicate that intrauterine position influences behaviors involved in the maintenance of metabolic homeostasis.     

Non-invasive measurement of adrenocortical activity in male and female rats

Rats are widely used in biomedical research as animal models for human diseases. However, due to their small body size, blood sampling is complicated and invasive and thereby can seriously interfere with endocrine functions and possibly compromise the animals' welfare. Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desired. Our study aimed to gain general information about corticosterone metabolism and excretion and to validate a 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay (EIA) to reliably measure faecal corticosterone metabolites (CMs) in laboratory rats. In total, 18 rats were administered 2.3 MBq of (3)H-corticosterone intravenously and per os, respectively (intravenous: 6 males and 6 females; per os: 3 males and 3 females). Subsequently, all voided excreta were frequently collected for five days. About 75+/-9% of the recovered CMs were found in the faeces. Peak concentrations of radiolabelled steroids appeared in the urine after 1.7+/-0.6 h in males and after 6.0+/-3.5 h in females. In faeces, maxima were observed after 14.7+/-2.4 h in both sexes. In principle, the time course and delay for both routes of administration (intravenous or per os) were the same, except for a delay of peak concentrations in urine (4.5+/-2.1 h) in per os administered males. Using high-performance liquid chromatography (HPLC), faecal (3)H-CMs were characterized and differences were found between the sexes. In both sexes, corticosterone was extensively metabolized, but while males showed only minor variations in their CM patterns, those of females differed largely between individuals. To validate the mentioned EIA, we investigated the diurnal variation (DV) of glucocorticoids as well as effects of the injection procedure itself and conducted an adrenocorticotropic hormone challenge test and a dexamethasone suppression test, using six male and six female rats each. Our results demonstrated that pharmacological stimulation, suppression and DV of adrenocortical activity were accurately reflected by means of CM measurement in faeces. By successful physiological validation, we proved for the first time the suitability of an immunoassay to non-invasively monitor adrenocortical activity in rats of both sexes. This method opens up new perspectives for biomedical and pharmacological investigations as well as for animal welfare related issues.     

Drug induction of hepatic glutathione S-transferases in male and female rats*.

The induction of the glutathione S-transferases by phenobarbital and polycyclic hydrocarbons was studied in male and female rats. Administration of phenobarbital resulted in 60-80% increase in S-aryl and S-aralkyl enzyme specific activities, whereas the S-epoxide and S-alkyl activities were increased by 30-40%. In following the sequence of induction, the former two activities were noted to reach peak activities before an increase in the latter two activities was observed. Both 3-methylcholanthrene and 3,4-benzopyrene were shown toi nduce these four enzymic activities, although without the discrimination between pairs of activities noted with phenobarbital. No change in Km accompanied the increase in Vmax. after induction by drugs, and no change occurred in Ki for sulphobromophthalein inhibition. Significantly lower enzyme specific activities were found for three of the activities studied in female rats but no difference was observed in the S-alkyltransferase activity. However, the proportional increase in the enzymic activities in response to phenobarbital was the same in males and females. These studies demonstrate the drug induction of a group of cytosolic drug-metabolizing enzymes as well as the identification of sex differences in these activities.     

Ingestion of soybean epoxide oil. Effects on monooxygenases, epoxide hydrolase and activities of UDP glucuronosyltransferases in hepatic microsomes of the rat

The effect of peroxidized soybean oil in the diet of male Wistar rats was studied on hepatic drug metabolizing enzymes and their phenobarbital induction and compared to that of natural soybean diet in the same conditions. No hepatomegaly or increase in serum transaminases occurred, however growth was inhibited after ingestion of peroxidized soybean oil. In addition, the protein biosynthesis of epoxide hydrase determined by immunochemistry was largely stimulated by this treatment; but the corresponding activity measured with benzo(a)pyrene 4-5 oxide as a substrate was increased in weaker proportions. This induction was limited to epoxide hydrolase only, since the enzymes of phase one were not affected and UDP glucuronosyltransferase activities toward group I substrates were randomly activated. The induction of epoxide hydrolase may affect only one or several isoforms of the membrane enzyme which are not necessarily specific to benzo(a)pyrene 4-5 oxide activity determination of the enzyme.     

Tolerance to acute ischemia in adult male and female spontaneously hypertensive rats

Clinical and experimental studies have repeatedly indicated that overloaded hearts have a higher vulnerability to ischemia/reperfusion injury. The aim of the present study was to answer the question whether the degree of tolerance to oxygen deprivation in hearts of spontaneously hypertensive rats (SHR) may be sex-dependent. For this purpose, adult SHR and their normotensive control Wistar Kyoto (WKY) rats were used. The isolated hearts were perfused according to Langendorff at constant pressure (proportionally adjusted to the blood pressure in vivo). Recovery of contractile parameters (left ventricular systolic, diastolic and developed pressure as well as the peak rate of developed pressure) was measured during reperfusion after 20 min of global no-flow ischemia in 5 min intervals. Mean arterial blood pressure was measured by direct puncture of carotid artery under light ether anesthesia in a separate group of animals. The degree of hypertension was comparable in both sexes of SHR. The recovery of contractile functions in SHR males and females was significantly lower than in WKY rats during the whole investigated period. There was no sex difference in the recovery of WKY animals; on the other hand, the recovery was significantly better in SHR females than in SHR males. It may be concluded that the hearts of female SHR are more resistant to ischemia/reperfusion injury as compared with male SHR. This fact could have important clinical implications for the treatment of cardiovascular disease in women.     

Species differences in cytochromes P-450 and epoxide hydrolase: comparisons of xenobiotic-induced hepatic microsomal polypeptides in hamsters and rats

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.