The effect of haem biosynthesis inhibitors and inducers on intestinal iron absorption and liver haem biosynthetic enzyme activities.

The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p<0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p<0.005, p<0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p<0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p<0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p<0.05, p<0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p<0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p<0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p<0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption.

Hemin-mediated restoration of allylisopropylacetamide-inactivated CYP2B1: a role for glutathione and GRP94 in the heme-protein assembly.

Administration of the cytochrome P450 (P450) suicide inactivator allylisopropylacetamide (AIA) to phenobarbital (PB)-pretreated rats results in rapid and marked inactivation of several liver endoplasmic reticulum (ER)-bound P450s. A few of these such as CYP2B1, inactivated due to AIA-mediated prosthetic heme N-alkylation, can be structurally and functionally restored nearly completely by exogenous hemin in vivo or in vitro. Such in vitro hemin-mediated reassembly is unsuccessful with purified AIA-inactivated CYP2B1 and, as shown herein, is not very effective even when heme is incubated with just the corresponding liver microsomes that contain the reconstitutable CYP2B1 protein, thereby implicating a requirement for additional factors provided by the intact liver cell homogenates, ER, and/or cytosol. Using various approaches that include high-performance liquid chromatographic fractionation of the liver cytosolic subfraction as well as chemical and immunological probes such as the Hsp90/GRP94-specific inhibitor geldanamycin (GA) and polyclonal anti-GRP94 antibodies, respectively, we now demonstrate that the in vitro hemin-mediated reassembly of heme-stripped microsomal CYP2B1 requires GSH as well as the ER chaperone GRP94, but not the cytosolic chaperone heat shock protein 90. It remains to be determined whether GSH acts directly or indirectly, via a putative ER thiol reductase, to maintain the conserved active site cysteine-thiol (Cys436 in CYP2B1) in a reduced state, competent for heme binding and repair.

Rhodanese and ALA-S in mammary tumor and liver from normal and tumor-bearing mice.

1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-amino-levulinate synthetase (ALA-S), cytoplasmic and mitochondrial rhodanese were determined in tumor (T) and liver of both normal mice (NM) and T-bearing mice (TBM). 2. Rhodanese tumoral mitochondrial levels were higher than the hepatic normal mitochondrial fraction, while the cytoplasmic activity was nearly equal in all sources. 3. In neither case was the activity of tumoral ALA-S and rhodanese altered by any of the porphyrinogenic drugs. 4. Mitochondrial and cytoplasmic rhodanese activity was also measured in tumor and liver of TBM at different intervals after transplantation. We concluded that the behaviour of rhodanese is a property inherent to the tissue and not one attained with time.

Metabolism and alkylating activity of thio-TEPA in rat liver slice incubation.

Precision-cut rat-liver slices were used to study the metabolism of the alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA). Exposure to high concentrations (1-10 mM) of thio-TEPA for 6 h did not prove to be toxic to the liver slices as indicated by insignificant leakage of potassium from the cells. The time course of the disappearance of thio-TEPA (initial concentration, 5.2 microM) from the buffer during incubation followed first-order kinetics. Formation of N,N'N''-triethylenephosphoramide (TEPA) apparently accounted for the elimination of thio-TEPA. Pretreatment of the rats with phenobarbital significantly increased the reaction rate. Conversely, pretreatment with the cytochrome P-450 inhibitor allylisopropylacetamide significantly reduced the metabolic rate. The elimination of thio-TEPA and formation of TEPA occurred independently of thio-TEPA concentration, which ranged from 5.2 to 104 microM. Thio-TEPA's oxo-analogue TEPA, which was not further metabolized, was the only metabolite identified. However, a significantly time-related increase in 4-(nitrobenzyl)-pyridine (NBP) alkylating activity was observed following incubation of liver slices with thio-TEPA but not after their incubation with TEPA. This may possibly indicate the formation of unknown active metabolites.

Studies on induction of delta-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin. Chlorpropamide, allylisopropylacetamide and lead in hepatocytes from normal and experimental diabetic rats.

The present work demonstrates that phenformin exerted an inducing effect on delta-aminolevulinic acid synthase (ALA-S) and ferrochelatase activities and on cytochrome P-450 content in isolated hepatocytes from rats with experimental diabetes. Similar results were obtained with respect to ALA-S activity and cytochrome P-450 content when chlorpropamide was used. The inducing effect exerted by allylisopropylacetamide (AIA) on ALA-S and ferrochelatase activities in diabetic hepatic cells was markedly greater than that observed in normal hepatocytes. This stimulatory response was not enhanced by adding dibutyryl cyclic AMP (cAMP). When phenformin was added to isolated rat hepatocytes of normal rats, induction of ALA-S and ferrochelatase activities and cytochrome P-450 content was observed only in the presence of added dibutyryl cAMP. Addition of chlorpropamide to this in vitro system did not exert an inducing effect on the same enzymes even in the presence of dibutyryl cAMP. The present results add more experimental evidence about the lability of the heme pathway of diabetic hepatocytes.

Heme regulation in mouse mammary carcinoma and liver of tumor bearing mice--I. Effect of allyl-isopropylacetamide and veronal on delta-aminolevulinate synthetase, cytochrome P-450 and cytochrome oxidase.

1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver.

Synergistic effect of allyl isopropyl acetamide with cadmium on hepatic heme synthesis in chick embryo.

The effect of cadmium along with a porphyrogenic drug, allyl isopropyl acetamide, on the induction of 5-amino levulinic acid (ALA) synthetase, ALA dehydratase and heme level was studied. The interaction of cadmium with allyl isopropyl acetamide indicated that the decrease in hepatic heme level by cadmium or allyl isopropyl acetamide may occur in a synergistic manner, whereas the induction of ALA synthetase by cadmium or allyl isopropyl acetamide may not take place in the same manner. Further, neither allyl isopropyl acetamide treatment alone nor allyl isopropyl acetamide-cadmium treatment had any effect on ALA dehydratase activity.

Structure-pharmacokinetic relationships in a series of valpromide derivatives with antiepileptic activity.

The following valpromide (VPD) derivatives were synthesized and their structure-pharmacokinetic relationships explored: ethylbutylacetamide (EBD), methylpentylacetamide (MPD), propylisopropylacetamide (PID), and propylallylacetamide (PAD). In addition, the anticonvulsant activity of these compounds was evaluated and compared to that of VPD, valnoctamide (VCD), and valproic acid (VPA). MPD, the least-branched compound had the largest clearance and shortest half-life of all the amides investigated and was the least active. All other amides had similar pharmacokinetic parameters. Unlike the other amides, PID and VCD did not metabolize to their respective homologous acids and were the most active compounds. Our study showed that these amides need an unsubstituted beta position in their aliphatic side chain in order to biotransform to their homologous acids. An amide which is not metabolized is more potent as an anticonvulsant than its biotransformed isomer. All amides were more active than their respective homologous acids. In this particular series of aliphatic amides, which were derived from short-branched fatty acids, the anticonvulsant activity was affected by the pharmacokinetics in general and by the biotransformation of the amide to its homologous acid in particular. This amide-acid biotransformation appeared to be dependent upon the chemical structure, especially upon the substitution at position beta of the molecule.

Role of xanthine oxidase in the interferon-mediated depression of the hepatic cytochrome P-450 system in mice.

Interferon, interferon inducers, and a variety of other immunomodulators are known to depress the hepatic cytochrome P-450 drug-metabolizing system. Two concepts have been proposed to explain this phenomenon. (a) The steady-state of cytochrome P-450 is altered through decreased synthesis and increased degradation of cytochrome P-450 apoprotein. (b) Interferon induces xanthine oxidase; superoxide generated by interferon-induced xanthine oxidase destroys cytochrome P-450. The current study investigated the second concept. Administered polyribonucleotides [polyriboinosinic acid.polyribocytidylic acid (poly IC), polyriboinosinic acid.polycytidylic acid, polylysine and carboxymethylcellulose, mismatched poly IC], recombinant murine gamma-interferon, and a natural murine alpha/beta-interferon were shown to depress hepatic cytochrome P-450 and selected microsomal cytochrome P-450-dependent monooxygenase reactions and to induce hepatic xanthine oxidase activity. The feeding of tungstate in the drinking water largely depleted xanthine oxidase in mice; cytochrome P-450 levels and monooxygenase activities were not affected by tungstate treatment. Tungstate rendered the level of xanthine oxidase much below that in mice that had not received tungstate regardless of whether or not they had received poly IC or interferon; nevertheless, poly IC and interferon produced losses of cytochrome P-450 and monooxygenase activities in these tungstate-treated mice equivalent to those observed in mice that had not received tungstate. The administration of N-acetylcysteine did not prevent the loss of cytochrome P-450 induced by poly IC, as has been reported, nor did the incubation of microsomal cytochrome P-450 with buttermilk xanthine oxidase and hypoxanthine cause a loss of cytochrome P-450, which has also been reported. It is concluded from these studies that the induction of xanthine oxidase and the loss of cytochrome P-450 generated by interferon are coincidental rather than causally related phenomena.

Effect of aliphatic amides on oncogenic transformation, sister chromatid exchanges, and mutations induced by cyclopenta[cd]-pyrene and benzo[a]pyrene.

We examined the effects of the aliphatic amides isopropyl-valeramide (IVA) and allylisopropylacetamide (AIA) on oncogenic transformation and sister chromatid exchanges (SCE) induced by cyclopenta[cd]pyrene (CPP) and benzo[a]pyrene (B[a]P) in C3H/10T1/2 cells and on B[a]Pdiol-epoxide (BPDE)-induced mutation at the HGPRT locus in Chinese hamster ovary (CHO) cells. IVA and AIA significantly suppressed B[a]P and CPP transformation in vitro. Both amides were effective when given just prior to, simultaneously with, or 24 h after carcinogen exposure. On the other hand, IVA and AIA did not affect cytotoxicity, the frequencies of SCE induced by CPP or B[a]P, nor BPDE-induced mutations in CHO cells. These and previous results suggest that the mechanism of inhibition of transformation by IVA or AIA may be very specific and probably not related to the early initiation event in oncogenic transformation in vitro.

The cDNA and protein sequence of a phenobarbital-induced chicken cytochrome P-450.

Several cDNA clones complementary to a chicken phenobarbital-inducible cytochrome P-450 have been isolated and sequenced, representing the first non-mammalian eukaryotic cytochrome P-450 sequence to be analyzed. The cDNA clones hybridized to two mRNAs of 3.5 and 2.5 kilobases in length, but further analysis indicated that the clones were derived from the larger mRNA. The sequence contains a 5'-noncoding region of 39 nucleotides and an open reading frame of 1473 nucleotides. The remainder of the sequence is due to the 3'-noncoding region and poly(A) tail. The open reading frame encodes a protein of 491 amino acids with a molecular weight of 56,196. The chicken cytochrome P-450 shows an overall homology of 45-54% compared with the mammalian phenobarbital-induced cytochrome P-450s. The degree of homology is not uniform, with some short regions showing much greater levels of sequence conservation. In particular, the chicken cytochrome P-450 contains the conserved cysteinyl domain near the carboxyl terminus, found in all cytochrome P-450s and which is thought to be involved in heme binding. Using the chicken sequence, a more accurate estimate of the evolutionary rates of cytochrome P-450s has been made. It is suggested that the phenobarbital-, 3-methylcholanthrene, and pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 gene families diverged from a common ancestral gene 600 million years ago. Furthermore the phenobarbital-inducible gene apparently underwent gene duplication events at about the time of the divergence of the chicken and mammalian lineages. The results imply that most mammals should have at least four rather distantly related phenobarbital-inducible gene subfamilies.

Influence of isopropylvaleramide and allylisopropylacetamide on transformation of C3H/10T1/2 cells induced by benzo[a]pyrene derivatives.

We examined the effect of aliphatic amides isopropylvaleramide (IVA) and allylisopropylacetamide (AIA) on the oncogenic transformation of C3H/10T1/2 cells induced by benzo[a]pyrene (B[a]P) or its proximate and ultimate metabolites (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (B[a]P-diol-epoxide), respectively. IVA and AIA given prior to, simultaneously with, or for 24 h intervals beginning up to 48 h after removal of carcinogens significantly suppressed transformation induced by B[a]P or the 7,8-diol metabolite. Both modifiers were most effective when added for 24 h immediately following carcinogen exposure. IVA and AIA were also very potent inhibitors of B[a]P-diol-epoxide transformation; however they were most effective when added for 24 h simultaneously with the B[a]P-diol-epoxide. No significant difference in B[a]P-diol-epoxide binding to DNA in C3H/10T1/2 cells was observed during 1 or 24 h exposure to this carcinogen in the presence or absence of IVA or AIA. Neither modifier affected X-ray transformation when added for 24 h immediately following X-irradiation of C3H/10T1/2 cells. These results suggest that AIA and especially IVA might be important tools in studies directed at non-metabolic aspects of B[a]P carcinogenesis.

Increased glutathione in cultured hepatocytes associated with induction of cytochrome P-450. Lack of effect of glutathione depletion on induction of cytochrome P-450 and delta-aminolevulinate synthase.

Cellular glutathione concentrations in primary cultures of chick embryo hepatocytes were 15.3 +/- 5.3 nmoles/mg protein (mean +/- S.D.) and remained stable for up to 3 days in culture. The presence of insulin was not essential for the maintenance of glutathione concentrations. Induction of cytochrome P-450 by phenobarbital-like inducers (2-propyl-2-isopropylacetamide, 2-allyl-2-isopropylacetamide, and 2,4,5,2',4',5'-hexabromobiphenyl) was accompanied by 2- to 3-fold increases in glutathione concentrations and by increased glucuronidation of phenol red. The 3-methylcholanthrene-like inducers of cytochrome P-450 (beta-naphthoflavone and 3,4,3',4'-tetrachlorobiphenyl) did not have these effects. Glutathione was rapidly depleted to 15-30% of control levels in hepatocytes treated with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthase. No toxicity was observed with glutathione depletion. Glutathione depletion did not affect the ability of 2-propyl-2-isopropylacetamide to induce cytochrome P-450, glucuronidation of phenol red, or delta-aminolevulinate synthase.

Effect of glucose on the induction of delta-aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide.

The present work shows that allylisopropylacetamide exerts an inducing effect on delta-aminolevulinic acid synthase and ferrochelatase activities in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the inducing effect produced in both enzymes. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase in this in vitro system. A similar effect was observed with fructose and 2-deoxyglucose. No glucose effect was observed with galactose, mannose, glycerol, pyruvate and lactate. The glucose effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. The simple in vitro method used in the present work promises to be a very useful tool for studies of regulatory mechanisms of porphyrin and heme biosynthesis in hepatocytes under normal and pathological conditions (hepatic porphyrias).

Metabolism of estradiol in liver cell culture. Differential responses of C-2 and C-16 oxidations to drugs and other chemicals that induce selective species of cytochrome P-450.

The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17 beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as the substrate. Oxidation of estradiol by the cultured liver cells was assessed by the release of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative reactions were detectable in the liver cell cultures by this technique. When incubated with a concentration of estradiol substrate close to the Michaelis constant (Km), approximately 45.8 pmol [2-3H]estradiol and 5.0 pmol [16-3H]estradiol/mg protein per minute underwent oxidative metabolism in untreated cells. Total amounts of oxidized product formation after 2 h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively. Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively), whereas no significant change in C-16 oxidation was observed after treatment of the cultures with 3-methylcholanthrene, benzo[a]pyrene, or benz[a]anthracene. The latter chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e., 1.5-2.2-fold increases over control values). The increase in C-2 oxidation after treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that observed for oxidation at C-16. The apparent Km values for these oxidations in control cultures were 23.5 and 30.3 microM for C-2 and C-16 oxidation, respectively; corresponding maximum velocity (Vmax) values were 119 and 11.7 pmol/mg protein per minute, respectively. These data indicate that the C-2 and C-16 oxidations of estradiol take place in cultured avian hepatocytes and that the extent of metabolism at these positions on the hormone molecule can be altered by chemicals, such as drugs and polycyclic aromatic hydrocarbons, which induce distinctive species of cytochrome P-450 in the liver.

Rat liver protoporphyrinogen IX oxidase: site of synthesis and factor influencing its activity.

Rat liver protoporphyrinogen IX oxidase is not formed in mitochondria in contrast to the claims made for the yeast enzyme (Poulson and Polglase, FEBS Lett. (1974) 40, 258). Inhibition of mitochondrial protein synthesis in regenerating rat livers by thiamphenicol led, instead, to a slight increase in protoporphyrinogen oxidase activity. Protoporphyrinogen IX oxidase was not induced in rat liver by triiodothyronine, an inducer of mitochondrial protein synthesis, or by AIA, an inducer of heme synthesis. Significant increases in activity were observed to be associated with rapidly growing cells, such as regenerating livers and rat ascites hepatoma cells.

Iron and the liver: acute effects of iron-loading on hepatic heme synthesis of rats. Role of decreased activity of 5-aminolevulinate dehydrase.

Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed by addition of dithiothreitol, Zn2+ or chelators of Fe2+ and Fe3+. Addition of FeSO4, ferric citrate, or ferritin to homogenates of control liver had no effect of activity of ALA dehydrase. The decrease in activity of ALA dehydrase, caused by iron-dextran, was mirrored by a reciprocal increase in ALA synthase. Iron-dextran potentiated the induction of ALA synthase by allylisopropylacetamide. However, this potentiation could be dissociated from the decrease in ALA dehydrase caused by iron loading.