Effect of lead on some parameters of the heme biosynthetic pathway in rat tissues in vivo

Lead was administered to male and female rats in drinking water for 3 and 6 weeks at the following doses: 0, 10, 100, 1000, 5000 ppm and for 6 months at 10 ppm only. Various parameters of blood-lead concentration (Pb-B), hematocrit (Htc), hemoglobin (Hb), free erythrocyte porphyrins (FEP), δ-aminolevulinate dehydratase (AlAD), reticulocytes- and tissu-ALAD, free tissue pophyrins (FTP), lead concentration (Pb-T)-were determined. Pb-B incrases with dose but reaches rapidly a plateau despite continuous Pb administration.Concentratoni of Pb in kidney, liver and brain correlates with Pb-B. Pb does not accumulate in heart. Kidney is the main site of Pb deposition and kidney ALAD is the parameter most susceptible to lead, since reduction is observed in all treated groups after 3 weeks of exposure.However, kidney ALAD inhibition in transitory since after 6 weeks it is only observed in the 5000 ppm group. At 10 ppm lead prevents also the increase in blood ALAD activity normally associated with the reticulocytosis of repetitive bleeding. The next parameters affected by lead are: ALAD in blood which in inhibited after 6 weeks of treatment with 100 ppm lead, and FEP, α-aminolevulinic acid plus other pyrrole-forming substances in urine (ALA-U), and FTP inkidney which are increased after 3 or 6 weeks of treatment with 100 and 5000 ppm lead.     

Environmental lead exposure induces changes in the heme biosynthetic pathway

Lead is ubiquitous in the environment today. Lead enters our body from a variety of sources such as urban environments and food. All humans have lead in their bodies primarily as a result of exposure to man-made sources. Children show a greater sensitivity to lead's effects than adults do. In this study, the concentration of metabolites of heme biosynthesis in school children from a group of volunteers with various blood lead contents and a group of lead-intoxicated children were reported. Also, the measurement of free erythrocyte porphyrins (FEP) as a microscreening test for lead toxicity was performed, and the blood lead levels of the school children were determined as well. The results show that the concentrations of the metabolites of heme biosynthesis are affected by the blood lead level. FEP level shows a small change as the blood lead level slightly increases. Elevation of blood lead level and the increase of the metabolite concentration are a good indication of lead-induced heme metabolic changes. FEP is an excellent screening test for the heme metabolic imbalances. Because of differences in individual susceptibility, symptoms of lead intoxication and their onset may vary. With increasing exposure, the severity of symptoms can be expected to change with varying degrees of lead toxicity. ©1997 by John Wiley & Sons, Inc. Environ Toxicol Water Qual 12 : 245–248, 1997     

The suitability of carbamazepine as a single-sample probe of human mixed function oxidase activity

1. Carbamazepine concentrations measured in plasma and plasma ultrafiltrates by fluorescence polarization immunoassay (f.p.i.a.) from 0 to 48 h post-dose were used to calculate CL, V, clearance of plasma unbound drug (CLunb), Vunb; mean (+/- SD) values for these were: 0.017 l/kg/h (+/- 0.004), 1.05 l/kg (+/- 0.14), 0.058 l/h/kg (+/- 0.012), and 4.28 l/kg (+/- 0.41), respectively. 2. A single-dose, single-sample procedure for estimating carbamazepine oral clearance was evaluated with a view to using carbamazepine as a probe in screening for host factor influences on human drug metabolism. Single sample estimates of carbamazepine clearance (CL) were closest to multiple sample values for carbamazepine clearance (CL) when blood samples were collected 48 h after carbamazepine ingestion. This was the case for plasma total carbamazepine and plasma unbound carbamazepine. 3. A value of 1.1 l/kg was used for V in calculating all single sample estimates of clearance (CL), and a value of 4.3 l/kg was used to calculate single sample estimates of clearance of plasma unbound drug (CLunb). The mean prediction error (MPE) was negatively biased for CL and CLunb values calculated from single carbamazepine concentrations in samples collected at 24, 36, and 48 h post-dose. MPE was less than 5% errant for CL and less than 1% errant for CLunb when those parameters were calculated from 48 h concentrations of plasma total carbamazepine or plasma unbound carbamazepine, respectively. Root mean squared error (r.m.s.e.) was lost lowest when 48 h post-dose samples were used for single-sample clearance estimates.     

Dietary ascorbic acid and hepatic mixed function oxidase activity in the guinea pig

Studies were carried out to characterize the response of hepatic mixed function oxidase (MFO) activity to chronic ascorbic acid deficiency and excessive ascorbic acid intake in the guinea pig. When guinea pigs were fed excessive ascorbic acid, there was a small increase in hepatic cytochrome P-450 which was unaccompanied by any alteration in drug-metabolizing enzyme activity. Similarly, induction of MFO activity by phenobarbital was not modified by excessive ascorbic acid administration. Chronic ascorbic acid deficiency resulted in depressed metabolism of aniline, aminopyrine, ethoxycoumarin and benzphetamine, but not of ethylmorphine, in comparison with animals fed diets containing control and/or excessive amounts of ascorbic acid. In contrast to the metabolism of all drugs studied, the 7 alpha-hydroxylation of cholesterol was depressed by both inadequate and excessive vitamin C intake, demonstrating the unique sensitivity of cholesterol 7 alpha-hydroxylase to dietary ascorbate.     

Altered activity of hepatic mixed function oxidase, cytochrome P-450 and glutathione-S-transferase by styrene in rat fetal liver.

The effect of in utero exposure to styrene 200 or 400 mg/kg/day orally was studied on mixed function oxidase activities, cytochrome P-450 and glutathione contents and on the glutathione-S-transferase activity in rat fetal liver. Activities of aminopyrene-N-demethylase, aniline hydroxylase, aryl hydrocarbon hydroxylase and the cytochrome P-450 contents were significantly decreased in the fetal liver. A significant decrease in the glutathione contents and the glutathione-S-transferase activity was also observed in the liver of the fetuses of styrene exposed animals. The current data show that prenatal exposure to styrene could adversely affect the developing biotransformation process.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.     

Inhibition of hepatic mixed function oxidase activity by propyl gallate

Propyl gallate was found to inhibit microsomal benzpyrene hydroxylase activity and demethylase activity with ethylmorphine, aminopyrine or benzphetamine as a substrate. The extent of inhibition with different substrates varied with the age and diet of the animals. The benzpyrene hydroxylase activity of the microsomes of the 3-methylcholanthrene-treated rats was shown to be less susceptible to propyl gallate inhibition. Propyl gallate does not inhibit the NADPH-dependent reduction of cytochrome P-450; therefore, the site of inhibition is not on NADPH-cytochrome c reductase as suggested previously. Propyl gallate interacts with cytochrome P-450 to produce a positive absorption peak around 420 nm, and it may also interfere with the binding of a type I substrate, benzphetamine. It inhibits ethylmorphine demethylation by a noncompetitive mechanism and aminopyrine demethylation by a mixed mechanism. The mode of propyl gallate inhibition and the implications of these observations are discussed.     

A cascade analysis of the interaction of mercury and coproporphyrinogen oxidase (CPOX) polymorphism on the heme biosynthetic pathway and porphyrin production

Mercury (Hg) exposure in various forms remains a persistent public health concern in many parts of the world. In previous studies, we have described a biomarker of mercury exposure characterized by increased urinary concentrations of specific porphyrins, pentacarboxyporphyrin (5-CP) and coproporphyrin (4-CP), and the atypical keto-isocoproporphyrin (KICP), based on selective interference with the fifth (uroporphyrinogen decarboxylase, UROD) and sixth (coproporphyrinogen oxidase, CPOX) enzymes of the heme biosynthetic pathway. Whereas this response occurs in a predictable manner among approximately 85% of subjects with Hg exposure, an atypical porphyrinogenic response (APR) has been observed in approximately 15% of Hg-exposed persons, in which the three porphyrins that are affected by Hg, i.e., 5-CP, 4-CP and, KICP, are excreted in substantial excess of that predicted on the basis of Hg exposure alone. This APR has been attributed to a specific polymorphism in exon 4 of the CPOX gene (CPOX4). In the present study, we sought to further confirm the hypothesis that the observed changes in porphyrin excretion patterns might serve as a biomarker of Hg exposure and potential toxicity by statistically modeling the cascading effects on porphyrin concentrations within the heme biosynthetic pathway of Hg exposure and CPOX4 polymorphism in a human population with long-term occupational exposure to elemental mercury. Our results are highly consistent with this hypothesis. After controlling for precursor porphyrin concentrations, we demonstrated that 5-CP and 4-CP are independently associated with Hg concentration, while KICP is associated only with the CPOX4. An unpredicted association of Hg with heptacarboxyporphyrin (7-CP) may indicate a previously unidentified point of mercury inhibition of UROD. These findings lend further support to the proposed utility of urinary porphyrin changes as a biomarker of exposure and potential toxicity in subjects with mercury exposure. Additionally, these findings demonstrate the successful application of a computational model for characterizing complex metabolic responses and interactions associated with both toxicant exposure and genetic variation in human subjects.     

Histamine inhibition of mixed function oxidase activity in rat and human liver microsomes and in the isolated perfused rat liver

The imidazole ring is a common structural feature of some xenobiotics that inhibit cytochrome P-450-catalysed reactions. Histamine is a 4-substituted imidazole and a preliminary study has shown it to be an inhibitor of rat liver microsomal drug oxidation. This work has now been extended. Histamine appears to be a competitive inhibitor of the alpha-hydroxylation (HM) (Ki = 164 microM; IC50 at 20 microM = 308 microM) and O-demethylation (ODM) (Ki = 243 microns; IC50 at 20 microM = 400 microM) of metoprolol in rat liver microsomes. Of the metabolites of histamine only N-acetylhistamine showed comparable inhibitory potency to that of the parent compound. Histamine impaired the disappearance of lignocaine when incubated with rat liver microsomes. This was accompanied by a corresponding inhibition of 3-hydroxy-lignocaine appearance. Histamine produced a type II spectral interaction with rat liver microsomes (lambda max = 432 nm, lambda min = 408 nm; Ks = 0.11 mM). When histamine was incubated alone with rat liver microsomes no loss of substrate was observed. The oxidation of metoprolol by human liver microsomes was impaired by histamine (IC50 values for ODM appearance at 25 microM: liver HL1 greater than 10, HL3 = 3.8 and HL4 = 3.7 mM). In comparison, cimetidine had an IC50 value of 1.5 mM using microsomes from liver HL3. Addition of histamine impaired the elimination of metoprolol by the isolated perfused rat liver in a dose-dependent manner (P less than 0.001, one-way analysis of variance). These data demonstrate that histamine can enter hepatocytes, interact with cytochrome P-450 and inhibit some drug oxidation reactions. The physiological relevance of inhibition of drug metabolism by histamine remains to be determined.     

Exposure to toxic agents: the heme biosynthetic pathway and hemoproteins as indicator

The heme biosynthetic pathway is closely controlled by levels of the end product of the pathway, namely, heme, and porphyrins are normally formed in only trace amounts. When control mechanisms are disturbed by xenobiotics, porphyrins accumulate and serve as a signal of the interaction between a xenobiotic and the heme biosynthetic pathway. For example, an increase in erythrocyte protoporphyrin is a useful measurement for early detection of exposure to lead and porphyrinuria was an early manifestation of a hexachlorobenzene-induced porphyria in Turkey. In recent years a variety of additional xenobiotics has been shown to interact with the heme biosynthetic pathway, namely, halogenated aromatic hydrocarbons, pesticides, sulfides, and a variety of metals. Moreover, different xenobiotics (e.g., dihydropyridines and compounds containing unsaturated carbon-carbon bonds) interact with the prosthetic heme of cytochrome P-450 forming novel N-alkylporphyrins.     

Effect of storage on microsomal mixed function oxidase activity in mouse liver

The stability of mixed function oxidase components from hepatic microsomes of mice was studied. Microsomal pellets were overlaid with isotonic, buffered KCl and frozen at ?20° for up to 20 days. Ethyl morphine N-demethylase and aniline hydroxylase were the least stable, showing significant decreases in activity after 3 and 5 days of storage respectively. NADPH cytochrome c reductase and cytochrome P-450 were both stable for at least 10 days. Electron micrographs of resuspended pellets after storage showed an increasing tendency toward aggregation of microsomal vesicles as the length of storage increased.     

Effect of acute ethanol administration on diamine oxidase activity in maternal, embryonal and fetal tissues

Pregnant rats were acutely treated with ethanol to study the influence of this drug on diamine oxidase activity of maternal, embryonal, and fetal tissues. When ethanol was given on day 12 of gestation, enzyme activity was unmodified in placenta and embryo, whereas it was reduced by 38 and 31%, respectively, in maternal liver and plasma at 3 h. When ethanol was given on day 18 of gestation, diamine oxidase activity diminished in maternal liver, plasma and placenta by about 35-40% at 6 h. Moreover, in the fetus ethanol caused a 35% diminution of enzyme activity in liver at 6 h and a 45% stimulation in brain at 3 h, and of about 65% at 6-12 h. These data may be of interest in view of the physiological role of diamine oxidase in the oxidation of the large amounts of amines produced during pregnancy.     

Effect of cimetidine or ranitidine pretreatment on hepatic mixed function oxidase activity in the rat

This study compared the effect of single equimolar oral doses of cimetidine (100 mg/kg) or ranitidine (139 mg/kg) on rat hepatic mixed function oxidases. Cimetidine significantly (p less than 0.05) increased hexobarbital sleeping time and prolonged aminopyrine and theophylline elimination. In contrast, ranitidine did not significantly affect hexobarbital sleeping time and theophylline elimination but significantly (p less than 0.025) increased aminopyrine elimination. Aminopyrine N-demethylase activity in vitro was significantly (p less than 0.05) inhibited by cimetidine pretreatment but significantly (p less than 0.025) increased by ranitidine pretreatment. The direct addition of cimetidine or SKF 525A to the 10,000g supernatant fraction from controlled liver homogenates decreased aminopyrine N-demethylase activity, whereas the direct addition of ranitidine tended to increase aminopyrine N-demethylase activity. A significant correlation (r = 0.65, p less than or equal to 0.005) was observed between hexobarbital sleeping time in vivo and aminopyrine N-demethylase activity in vitro in the same rat. The results of this study showed that cimetidine inhibited mixed function oxidases, whereas ranitidine had no effect or tended to stimulate mixed function oxidases.     

Specificity of medicarpin and related isoflavonoids in inhibition of rat hepatic mixed function oxidase activity

The cytochromes P-450 of the mixed function oxidase system metabolize a wide variety of endogenous compounds to either nontoxic products or toxic metabolites. A number of natural products, such as flavonoids, influence this metabolism. Exposure to these compounds may therefore be a factor in animal and human responsiveness to cytochrome P-450 substrates. We have examined the effect of the pterocarpan medicarpin on the cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin deethylase (ECD) activities of rat liver microsomes. Medicarpin and maackiain and two of their biosynthetic precursors inhibit the constitutive and phenobarbital (PB)-induced types of AHH, but have little effect on the 3-methylcholanthrene (MC)-induced type of AHH. This is in contrast to the effect of the commonly used cytochrome P-450 inhibitor 7,8-benzoflavone, which inhibits the hepatic AHH of MC-treated rats and has no effects on the AHH of control or PB-treated rats. However, medicarpin inhibited the constitutive as well as the PB- and MC-induced ECD. The specific modulatory effect as well as its relative availability suggests the utility of medicarpin as a probe for different forms of cytochrome P-450 in animal tissues.     

Polyamine oxidase activity in lymphoid tissues of glucocorticoid-treated rats

Glucocorticoids are known to negatively affect lymphoid tissues, in which they cause programmed cell death. Polyamine depletion, which occurs in glucocorticoid-treated animals by inhibition of biosynthesis and induction of acetylation, may represent a signal to thymocytes for progression into the apoptotic program. Since catalysis of polyamines by the catabolic pathway produces hydrogen peroxide as a by-product, it has been suggested that the apoptotic process may be, in part, due to oxidative stress as a result of hydrogen peroxide production. In order to verify whether polyamine oxidase (EC 1.5.3.11) may play a role in the process, we examined the activity of the enzyme in the thymus and spleen of glucocorticoid-treated rats. We administered dexamethasone (4 mg/kg) or two different doses of corticosterone (4 mg/kg or 30 mg/kg) to rats, which were killed 8 or 24 hr after hormone injection. We found that corticosterone and dexamethasone affected polyamine oxidase activity in both tissues, with an opposite dose-dependent effect of the natural hormone in the thymus. The decrease and increase in polyamine oxidase after the two doses of corticosterone were correlated with the absence and the occurrence of DNA fragmentation, respectively. Moreover, corticosterone affected polyamine oxidase activity earlier (8 hr) than dexamethasone (24 hr), but the synthetic hormone was more efficient than the natural hormone in thymic polyamine depletion. The polyamine oxidase response may represent an important event in lymphoid tissues after glucocorticoid treatment, suggesting a role of the enzyme in the catabolic effects exerted by the two hormones.