Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.     

Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.     

Selective induction of heme oxygenase-1 isozyme in rat testis by human chorionic gonadotropin

A radioimmunoassay was developed to assess the response of testicular HO-1 to agents known to increase the microsomal heme oxygenase activity. Treatment of rats with human chorionic gonadotropin (hCG) increased the microsomal heme oxygenase activity in rat testis. The following data suggest that the increase was specific to the HO-1 isozyme: (a) The elution profile of heme oxygenase activity from a DEAE-Sephacel column showed an increase in the HO-1 peak, but not in the HO-2 peak, (b) the Western immunoblot of the testis microsomes showed an increase in HO-1 protein, and (c) the amount of HO-1 protein that was present in the microsomes, when measured by radioimmunoassay, was doubled. Using radioimmunoassay, it was shown that other agents known to increase the testicular heme oxygenase, sodium arsenate and sodium arsenite, also increased the microsomal content of HO-1. An inhibitor of the testicular microsomal heme oxygenase activity, cadmium, also increased the microsomal HO-1 protein. The findings suggest that inducibility of HO-1 extends to tissues other than the liver, in this instance, the testis, and further support the possibility that HO-1 is the only inducible form of heme oxygenase.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats.

The effects of retinoic acid (RA) (50 micrograms/100 g body wt. per day) on hepatic heme oxygenase activity, delta-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T3 (10 micrograms/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P less than 0.01) and microsomal heme content by 47% (P less than 0.001). T3 administration enhanced heme oxygenase activity by 72% (P less than 0.001) and ALAS activity by 251% (P less than 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P less than 0.001) and heme levels by 75% (P less than 0.001). When RA and T3 were administered together, the retinoid markedly enhanced the T3 stimulation of heme oxygenase activity; 173% above controls (P less than 0.001), and 61% above T3 alone (P less than 0.001). However, RA failed to influence the effect of T3 on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

Effect of human chorionic gonadotropin and estradiol in vivo on estradiol binder and cytochrome P-450 concentrations in rat testis

The effects of single administration to adult male rats in vivo of various amounts of human chorionic gonadotropin (HCG) and of single or repeated injections of estradiol on testicular cytoplasmic estradiol binder concentrations and on microsomal progesterone-binding cytochrome P-450 were compared. Half-life periods of HCG-induced loss of estradiol binder and cytochrome P-450 concentrations are identical (6 h) whereas a strong dissociation of these half-life periods are evident after chronic estradiol treatment (less than 2 h for the estradiol binder, about 35 h for cytochrome P-450). Depletion of cytoplasmic estradiol binder is not a sufficient condition for mediation of effects on cytochrome P-450 content. Rate of replenishment of microsomal cytochrome P-450 is similar after HCG or estradiol treatment. Both HCG- and estradiol-induced loss of cytochrome P-450 occur not only in Leydig cells but also in microsomes prepared from seminiferous tubules. Additional information is presented contradicting the hypothesis that loss of cytochrome P-450-dependent steroidogenic enzymes caused by HCG could be mediated by estrogens.     

Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal

The treatment of male rats with Hg2+ resulted in significant alterations in heme and hemoprotein metabolism in the adrenal gland which, in turn, were reflected in abnormal steroidogenic activities and steroid output. Twenty-four hours after the administration of 30 mumol of HgCl2/kg (sc) the mitochondrial heme and cytochrome P-450 concentrations increased by approximately 50%. Also, Hg2+ treatment stimulated a porphyrinogenic response which included an 11-fold increase in the activity of delta-aminolevulinate synthetase. The increase in mitochondrial cytochrome P-450 content was reflected in elevated steroid 11 beta-hydroxylase and cholesterol side-chain cleavage activities. In contrast, Hg2+ treatment resulted in decreased concentrations of microsomal cytochrome P-450 (-75%) and heme (-45%). Similarly, the reduction in the microsomal cytochrome P-450 content was accompanied by reduced steroid 21 alpha-hydroxylase and benzo[alpha]pyrene hydroxylase activities. The mechanisms responsible for the loss of the microsomal cytochrome P-450 content appeared to involve a selective impairment of formation of the holocytochrome as well as an enhanced rate of heme degradation. This suggestion is made on the basis of findings that (a) the decrease in the microsomal cytochrome P-450 content was accompanied by a sevenfold increase in the activity of adrenal heme oxygenase, (b) no decrease in apocytochrome P-450 could be detected in sodium dodecyl sulfate-gel electrophoresis of the solubilized microsomal fractions stained for heme, and (c) the concentration of adrenal microsomal cytochrome b5 was significantly increased in the Hg2+-treated animals. It is suggested that Hg2+ directly caused a defect in adrenal steroid biosynthesis by inhibiting the activity of 21 alpha-hydroxylase. The apparent physiological consequences of this effect included lowered plasma levels of corticosterone and elevated concentrations of progesterone and dehydroepiandrosterone. This abnormal plasma steroid profile is indicative of a 21 alpha-hydroxylase impairment.     

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.     

The role of heme in the regulation of tryptophan-2,3-dioxygenase activity and content of cytochrome P-450 in rat liver

The effects of exogenous heme on the activity of delta-aminolevulinate synthase, heme oxygenase, tryptophan-2.3-dioxygenase and microsomal cytochrome content in rat liver were studied. It was shown that hemin chloride diminishes the delta-aminolevulinate synthase activity and provokes heme oxygenase induction. This is paralleled with the induction of the tryptophan 2.3-dioxygenase apoenzyme and an increase in the saturation of the enzyme with heme. The cytochrome b5 content does not change thereby, whereas that of cytochrome P-450 shows a decrease. Upon combined administration of actinomycin D and hemin the cytochrome P-450 level is markedly increased. Actinomycin D by itself has no effect on the hemoprotein concentration. It is concluded that the increase in the cytochrome P-450 level results from the activation of heme-induced mRNA translation.     

Tin-protoporphyrin inhibits heme oxygenase and prevents the decline in hepatic heme and cytochrome P-450 contents produced in nude mice by tumor transplantation

Heme and hemeprotein perturbations are present in nude mice bearing transplanted tumors. Hepatic microsomal heme oxygenase activity is increased 50-100% in tumor bearing nu/nu mice when compared with normal controls. This elevation in activity of the rate-limiting enzyme of heme degradation is associated with a 50% depletion of microsomal heme and cytochrome P-45 concentrations in liver. The synthetic heme analogue, Sn-protoporphyrin, a potent inhibitor of heme oxygenase, lowers the activity of heme oxygenase in tumor bearing animals to below control levels. This effect is associated with a normalization of hepatic heme and cytochrome P-450 contents. These findings might have implications for protecting normal cells during tumor growth and chemotherapy.     

Differential response of testicular and ovarian heme oxygenase activity to metal ions

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.     

Cadmium-mediated inhibition of testicular heme oxygenase activity: the role of NADPH-cytochrome c (P-450) reductase

The concerted activity of two microsomal enzymes, heme oxygenase and NADPH-cytochrome c (P-450) reductase, is required for isomer-specific oxidation of heme molecule; heme oxygenase is commonly believed to be rate limiting in this activity. In this report, we provide evidence strongly suggesting the rate-limiting role of the reductase in oxidation of heme molecule in rat testis. In the testis and the liver of rats treated with Cd (20 mumol/kg, sc, 24 h) heme oxygenase activity, assessed by the formation of bilirubin, was decreased by 50% and increased by 7-fold, respectively. In these animals, the reductase activity was decreased by nearly 75% in the testis, but remained unchanged in the liver. Similarly, the reductase activity in the liver was not altered when heme oxygenase activity was increased by 20-fold in response to bromobenzene treatment. Addition of purified testicular reductase preparation (purified over 4000-fold), or hepatic reductase, to the testicular microsomes of Cd-treated rats obliterated the Cd-mediated inhibition of heme oxygenase activity. The chromatographic separation of heme oxygenase and the reductase of the testicular microsomal fractions revealed that the reductase activity was markedly decreased (75%) while the heme oxygenase activity, when assessed in the presence of exogenous reductase, was not affected by in vivo Cd treatment. In vitro, the membrane-bound reductase preparation obtained from the testis was more sensitive to the inhibitory effect of Cd than the liver preparation. However, the purified reductase preparations from the testis and the liver exhibited a similar degree of sensitivity to Cd. Based on the molar ratio of heme oxygenase to the reductase in the microsomal membranes of the liver and the testis it appeared that the testicular heme oxygenase, which is predominantly HO-2 isoform, interacts with the reductase less effectively than HO-1; in the induced liver, heme oxygenase is predominantly the HO-1 isoform. It is suggested that due to the low abundance of NADPH-cytochrome c (P-450) reductase and the apparently lower affinity of the enzyme for HO-2, the reductase exerts a regulatory action on heme oxygenase activity in the testis.     

Regulation of heme and drug metabolism activities in the brain by manganese

A novel effect of metal ions in the brain is described. Mn was found to alter heme metabolism and the cytochrome P-450-dependent mixed-function oxidase activities in rat brain. A more than 2-fold increase in benzo(alpha)pyrene hydroxylase and 7-ethoxycoumarin deethylase activities were observed in the brain of rats treated for 7 days with Mn. The increases were regionally distributed; the highest elevations were observed in the hippocampus, pons and the caudate putamen. Moreover, in rats treated with Mn for 1 or 7 days a marked depression in the activity of the mitochondrial ALA synthetase was observed. The activity of the microsomal heme oxygenase was also inhibited at 7 days, but not 1 day, after Mn treatment. These inhibitions were reflected in an initial decrease, followed by a rebound return to normal, in the concentration of cytochrome P-450 in the brain. Mn was ineffective in vitro in altering heme and drug metabolism activities. It is suggested that Mn-mediated alterations in heme metabolic activities promote changes in the composition of cytochrome P-450 species in the brain microsomal fractions, such that the relative concentrations of the molecular species which catalyse aryl hydrocarbon hydroxylase activity become selectively increased.     

Activity of key enzymes of heme metabolism and cytochrome P-450 content in the rat liver in experimental rhabdomyolysis and hemolytic anemia

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.     

Depressed guinea pig testicular microsomal cytochrome P-450 content by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can result in hirsutism and chloracne, symptoms that suggest an alteration in endocrine regulation. Consequently, the effect of TCDD on guinea pig testicular cytochrome P-450 has been investigated. Twelve hours after a single, oral dose of TCDD (1 μg/kg), testicular microsomal cytochrome P-450 content was depressed by approximately 36%. Microsomal cytochrome P-450 content reached a maximal depression at approximately 1 day (52% of control) and remained at this level for 9 days. No appreciable alterations of testicular microsomal heme levels or activity of testicular δ-aminolevulinic acid (ALA) synthetase were observed.     

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of $\text{C57B1}\text{6}$ mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats

The effects of retinoic acid (RA) (50 μg/100 g body wt. per day) on hepativ heme oxygenase activty, δ-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T₃ (10 μg/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P < 0.01) and microsomal heme content by 47% (P < 0.001). T₃ administration enhanced heme oxygenase activity by 72% (P < 0.001) and ALAS activity by 251% (P < 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P < 0.001) and heme levels by 75% (P < 0.001). When RA and T₃ were administered together, the retinoid markedly enhanced the T₃ stimulation of heme oxygenase activity; 173% above controls (P < 0.001), and 61% above T₃ alone (P < 0.001). However, RA failed to influence the effect of T₃ on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

Detection of two heme oxygenase isoforms in the human testis

This study shows heme oxygenase multiplicity is common to rat and human tissues. The isozymes in man and rat, however, are heterogenous proteins that share certain characteristics. Two forms of heme oxygenase, HO-1 and HO-2, were identified in human testis. HO-2 form was the prevalent form. Human and rat HO-1 differed in chromatographic behavior and molecular weight; human HO-1 was a larger molecule (35,400 vs 30,000). The two forms, however, were similar in that immunochemically human HO-1 exhibited reactivity toward antibody to rat HO-1. Human and rat HO-2 also were dissimilar in chromatographic behavior and showed only a weak immunological cross-reactivity. Human and rat HO-1 were essentially the same size. As in rat organs, the microsomal cytochrome P-450 content in human testis was reciprocal to heme oxygenase activity.