Δ-aminolevulinic Acid Synthetase Activity Research Articles

Therapeutic effects of Moringa oleifera on arsenic-induced toxicity in rats

Moringa oleifera Lamarack (English: Horseradish-tree, Drumstick-tree; Hindi: Saijan; Sanskrit: Shigru) belongs to the Moringaceae family, is generally known in the developing world as a vegetable, a medicinal plant and a source of vegetable oil. Besides, the plant is reported to have various biological activities, including hypocholesterolemic agent, regulation of thyroid hormone status, anti-diabetic agent, gastric ulcers, anti-tumor agent and hypotensive agent, used for treating various diseases such as inflammation, cardiovascular and liver diseases. Therapeutic efficacy of oral administration of seed powder of M. oleifera (500 mg/kg, orally, once daily) post arsenic exposure (100 ppm in drinking water for 4 months) was investigated in rats. Animals exposed to arsenic(III) showed a significant inhibition of δ-aminolevulinic acid dehydratase (ALAD) activity, decrease in reduced glutathione (GSH) level and an increase in reactive oxygen species (ROS) in blood. On the other hand, a significant decrease in hepatic ALAD, and an increase in δ-aminolevulinic acid synthetase (ALAS) activity was noted after arsenic exposure. These changes were accompanied by an increase in thiobarbiturc acid reactive substances (TBARS) level in liver and kidney. Activities of liver, kidney and brain superoxide dismutase (SOD) and catalase also showed a decrease on arsenic exposure. Administration of M. oleifera seed powder post arsenic exposure, exhibited significant recovery in blood ALAD activity while, it restored blood GSH and ROS levels. Most of the other blood biochemical variables remained unchanged on M. oleifera supplementation. A significant protection in the altered ALAD and ALAS activities of liver and TBARS level in liver and kidney was however, observed after M. oleifera administration. Interestingly, there was a marginal but significant depletion of arsenic from blood, liver and kidneys. The results, thus lead us to conclude that post arsenic exposure administration with the seed powder of M. oleifera has significant role in protecting animals from arsenic-induced oxidative stress and in the depletion of arsenic concentration. Further studies thus can be recommended for determining the effect of co-administrating seed powder of M. oleifera during chelation therapy with a thiol chelator.

Glutathione depletion and anaesthesia in mice alter heme and drug metabolising enzymes

The effects of enflurane and isoflurane on heme metabolism, its regulation, and on some parameters involved in the hepatic drug metabolising system in animals under GSH depletion were investigated. A single dose of the anaesthethics (1 ml kg(-1), i.p.) was administered to control and GSH depleted mice, animals were sacrificed 20 min after. As a consequence of GSH depletion, a significant inhibition in delta-Aminolevulinic acid synthetase activity, the first enzyme of heme biosynthesis, and a striking induction in Heme oxygenase activity, the main enzyme of heme metabolism, were observed. Cytochrome P-450 levels and the activities of P-4502E1 and glutathione S-transferase were increased. These changes in heme metabolism and drug metabolising enzyme system were not altered further by the administration of enflurane or isoflurane. These findings would indicate that the status of oxidative stress produced by GSH depletion could not be affected by these anaesthetics and/or that disturbances in heme metabolism were already too important to undergo further variations.

Coordinate up-regulation of CYP1A1 and heme oxygenase-1 (HO-1) expression and modulation of δ-aminolevulinic acid synthase and tryptophan pyrrolase activities in pyridine-treated rats

To determine the changes in heme metabolism associated with induction of cytochrome P450 expression by pyridine, we compared the time course of CYP1A expression with the time course of (i) expression of heme oxygenase-1 (HO-1) (EC 1.14.99.3), (ii) activity of δ-aminolevulinic acid synthetase (ALAS) (EC 2.3.1.37), and (iii) heme saturation of tryptophan pyrrolase (TPO) (EC 1.13.11.11) in tissues of rats administered a single 100 or 150 mg/kg i.p. dose of pyridine. Both mRNA and protein of HO-1 and CYP1A1 were induced in the liver, kidney, and lung, with the induction of HO-1 mRNA preceding and paralleling that of CYP1A1 mRNA in the liver and lung but not kidney. Induction of CYP1A1 mRNA expression peaked within 9–12 hr and returned to control levels by 24 hr in all tissues examined, whereas induction of HO-1 mRNA expression was sustained for 48 hr in the lung and liver. In contrast to the transient up-regulation of CYP1A1 mRNA, increased microsomal CYP1A1 protein was sustained in all three tissues. Similar to the induction of HO-1 expression, lipid peroxidation was stimulated by pyridine treatment in the kidney, lung, and liver, but with the stimulation being more persistent in the liver and lung than in the kidney. Increased hepatic CYP1A1 or CYP1A2 activity was preceded by increased activities of HO-1 and ALAS. Pyridine treatment negatively modulated heme saturation of hepatic TPO. The findings indicate that pyridine stimulates the synthesis, utilization, and degradation of heme in a coordinate manner, and suggest that these alterations in heme metabolism may contribute to CYP1A1 induction by pyridine.

Effect of Voluntary Resistance Exercise on Mitochondrial Heme Biosynthesis in Rat Liver.

To evaluate the effects of voluntary resistance exercise training on mitochondrial heme biosynthesis in the liver, we trained adult male Sprague-Dawley rats for 4 or 8 weeks to climb a wire-mesh tower (∅20cm×200cm), and then measured δ-aminolevulinic acid synthetase (ALAS) activity, the rate-limiting enzyme of heme synthesis, and cytochrome c content in their liver. The activity of citrate synthase in the skeletal muscle was also determined to establish whether the training protocol had any aerobic effect. Resistance training resulted in significant increases (p<0.05) in citrate synthase activities in gastrocnemius and forearm muscles, whereas ALAS activity and cytochrome c content in the liver of rats trained for 4 and 8 weeks were the same as those of corresponding sedentary rats. The relative weight of forearm muscle to body mass was significantly greater (p<0.05) in the training group than in the sedentary group. These results suggest that voluntary resistance exercise does not accelerate hepatic heme biosynthesis in spite of increasing aerobic capacity in the skeletal muscles.

Altered Heme Pathway Regulation and Drug Metabolizing Enzyme System in a Mouse Model of Hepatocarcinogenesis: Effect of Veronal

1. Male CF 1 mice were fed p-dimethylaminoazobenzene (DAB) for 35 days and received 5,5-diethylbarbituric acid, before or after DAB treatment, with the purpose of investigating whether the onset of the preinitiation stage of carcinogenesis alters the natural regulatory mechanism of the heme pathway. 2. Changes detected in drug metabolizing enzymes are likely to be the consequence of a primary deregulation mechanism of heme metabolism, shown by an increase in δ-aminolevulinic acid synthetase activity and a decrease in microsomal heme oxygenase, which would finally lead to a great enhancement of cytochrome P450 levels. 3. The alterations found here would give rise to a pattern distinctive to that usually observed in the so-called resistant hepatocyte.

STZ-induced diabetes in mice and heme pathway enzymes. Effect of allylisopropylacetamide and α-tocopherol

A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of α-tocopherol (α-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic δ-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. α-Tocopherol did not modify ALA-S activity in either group. δ-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. α-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but α-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of a-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.

Changes of Hepatic Microsomal Oxidative Drug Metabolizing Enzymes in Chronic Renal Failure (CRF) Rats by Partial Nephrectomy

Male SD rats, 7-weeks-old, were used to investigate the changes in the hepatic drug metabolizing system of chronic renal failure (CRF) model rats. Partial nephrectomy (5/6) was performed in a two-stage surgical procedure. After nephrectomy, the rats were housed under regular conditions at least 21 days. After confirming the CRF states, trimethadione (TMO, 100 mg/kg, i.p.) was administered for evaluation of the hepatic drug metabolizing capacity; the ratio of dimethadione (DMO: the only metabolite of TMO) to TMO (DMO/TMO) in the serum and the dialysate from the blood microdialysis method were ascertained. The hepatic drug metabolizing enzyme contents and activities were also determined. In the CRF rats, the DMO/TMO ratios decreased significantly; total cytochrome P450 (CYP) contents, aminopyrine N-demethylase activity and δ-aminolevulinic acid synthetase activity also decreased significantly in the CRF rats. The extent of the alterations of these enzyme contents and activities correlated well with the severity of the CRF states evaluated by the serum blood urea nitrogen and creatinine concentrations. With Western blot analysis, the levels of CYP2C6, CYP2C11 and CYP3A2 decreased considerably in the CRF rats. These results suggest that CRF states induce not only a reduction of renal function but also an alteration of hepatic metabolism.

Effect of multiple doses of volatile anesthetics on heme enzymes

1. The effect of the administration of several doses of Enflurane and Isoflurane (2 ml/kg, i.p., daily) on heme metabolism and glucose levels was studied. 2. Liver and kidney δ-Aminolevulinic acid synthetase (ALA-S) activities were 85% ( P < 0.01) induced after the third dose of Enflurane, instead induction of this enzyme was only detected, in animals receiving one dose of Isoflurane. 3. Blood Porphobilinogenase and deaminase (50%, P < 0.01) inhibition was produced only when animals received a single dose of the anesthetics. 5. ALA-S induction observed after the third dose of anesthetics could be a consequence of long lasting depletion in heme synthesis produced by blocking at uroporphyrinogen level.

Alterations in hepatic δ-aminolevulinic acid synthetase and heme oxygenase activities after chronic ethanol consumption in rats

In the present study, the effect of chronic ethanol consumption in rats on the hepatic heme metabolism was investigated. Male Wistar rats were fed a nutritionally adequate liquid diet containing ethanol as 36% of the total calories for 5 weeks. After an overnight fast, the livers were excised and centrifuged to obtain mitochondrial and microsomal fractions. Chronic ethanol feeding of rats resulted in about 19% hepatomegaly as represented by the increased liver/body weight ratio. There was no difference in the mitochondrial protein content between the ethanol-treated and control rats, but the microsomal protein content was significantly increased in the ethanol-treated rats. Hepatic microsomal content of cytochrome P-450 (P-450) was markedly enhanced by chronic ethanol ingestion. Microsomal contents of cytochrome b5 (b5) and total heme were also increased to a lesser extent. After chronic ethanol abuse, the hepatic activity of delta-aminolevulinic acid (ALA) synthetase, which is a rate-limiting enzyme for heme production, was significantly increased and that of the heme oxygenase was slightly increased. These data indicate that ALA synthetase activity is induced by the negative feedback mechanism in order to compensate the depletion of heme caused by the utilization of heme for P-450. It is also speculated that, in response to excessive production of heme as described above, heme oxygenase activity is secondarily induced to regulate the amount of heme.

STUDY ON ANEMIA IN PATIENTS WITH RHEUMATOID ARTHRITIS

To elucidate the mechanism of anemia in rheumatoid arthritis (RA), iron-related parameters were examined in 26 RA patients, and ferrokinetic and erythrokinetic studies were performed on 9 and 8 of the 26 cases, respectively. As a whole, mild or moderate hypochromic normocytic anemia was observed. Serum iron levels were markedly decreased and the percent transferrin iron saturation was decreased or normal, while the serum concentration of ferritin fell within the normal range in all but one of the cases examined. Ferrokinetic studies revealed a decreased red cell iron turnover rate in 4 out of 9 cases and reduced red cell iron utilization in 2 out of 9 cases. Erythrokinetic studies disclosed that red cell life span was shortened in 5 cases and that total red cell volume was decreased in all 8 cases examined. Hemoglobin production per day was reduced in all 8 cases. In addition, the activity of δ-aminolevulinic acid synthetase was decreased in 2 cases examined. These findings suggest that abnormal iron metabolism and heme synthesis may be involved as causes of the anemia complicating RA.

Distribution of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase in liver and kidney of rainbow trout ( Salmo gairdnerii)

1. 1. Activities of δ-aminolevulinic acid synthetase (ALA-S) and δ-aminolevulinic acid dehydratase (ALA-D) in trout liver and kidney were compared with those in the mouse. 2. 2. ALA-S activity (per unit tissue fresh weight) exceeded ALA-D activity in trout liver and kidney. 3. 3. In trout kidney, ALA-S activity slightly exceeded, and ALA-D activity far exceeded, their activities in trout liver. 4. 4. In trout, heme synthesis differs from that in mammals in that appreciable synthesis occurs in the kidney, and in that ALA-S activity in not rate limiting.

Acute Effect of Amitriptyline, Phenobarbital or Cobaltous Chloride on δ-Aminolevulinic Acid Synthetase, Heme Oxygenase and Microsomal Heme Content and Drug Metabolism in Rat Liver

Cobaltous chloride (CoCI2) caused very marked decreases of cytochrome P-450, b5 and total heme contents and an increase of heme oxygenase activity. On the contrary, phenobarbital (PB) increased hepatic drug-metabolizing enzymes, but the total heme content remained unchanged. On the other hand, amitriptyline (AMT) caused a marked increase of δ-aminolevulinic acid (δ-ALA) synthetase activity at 12 and 24 hr. In addition, the contents of total heme and cytochrome b5 and the activities of aminopyrine (AM) N-demethylase and aniline (AN) hydroxylase at 24 hr were also increased by AMT, whereas cytochrome P-450 content did not change. This may be explained by the fact that AMT would increase hepatic heme synthesis through the prolonged induction of d-ALA synthetase, but it may not cause an increase in cytochrome P-450 heme because there are increases in the contents of cytochrome b5 and total heme.

Acute effect of amitriptyline, phenobarbital or cobaltous chloride on delta-aminolevulinic acid synthetase, heme oxygenase and microsomal heme content and drug metabolism in rat liver.

Cobaltous chloride (CoCl2) caused very marked decreases of cytochrome P-450, b5 and total heme contents and an increase of heme oxygenase activity. On the contrary, phenobarbital (PB) increased hepatic drug-metabolizing enzymes, but the total heme content remained unchanged. On the other hand, amitriptyline (AMT) caused a marked increase of delta-aminolevulinic acid (delta-ALA) synthetase activity at 12 and 24 hr. In addition, the contents of total heme and cytochrome b5 and the activities of aminopyrine (AM) N-demethylase and aniline (AN) hydroxylase at 24 hr were also increased by AMT, whereas cytochrome P-450 content did not change. This may be explained by the fact that AMT would increase hepatic heme synthesis through the prolonged induction of delta-ALA synthetase, but it may not cause an increase in cytochrome P-450 heme because there are increases in the contents of cytochrome b5 and total heme.

Contribution of methylsulfonyl metabolites of m-dichlorobenzene to the heme metabolic enzyme induction by the parent compound in rat liver

In the present study, we investigated the contribution of methylsulfonyl metabolites derived from m-dichlorobenzene ( m-DCB) on the heme metabolic enzyme induction by the parent compound in rats. The time courses of the effects of a single ip administration of m-DCB (200 mg/kg, 1.36 mmol/kg) and 2,4- and 3,5-dichlorophenyl methyl sulfones (2,4- and 3,5-DCPSO 2Mes) (each 50 μmol/kg) on hepatic microsomal cytochrome P450 content were almost in parallel with those on the total heme content in liver microsomes. m-DCB significantly increased the heme oxygenase activity, but 2,4- and 3,5-DCPSO 2Mes did not. On the other hand, m-DCB and both methyl sulfones markedly enhanced the δ-aminolevulinic acid (ALA) synthetase activity. No change was observed in percentage saturation of the tryptophan pyrrolase activity after administration of m-DCB, whereas this ratio at 6 hr after injection of 3,5-DCPSO 2Me was increased. In the liver of the dl-buthionine-( S,R)-sulfoximine (BSO)-treated rats dosed with m-DCB, both of 2,4- and 3,5-DCPSO 2Mes were present at significantly lower concentrations than in non-BSO-treated rats. Additionally, the m-DCB did not elevate the ALA synthetase activity in the BSO-treated rat. On the other hand, the administration of either 2,4- or 3,5-DCPSO 2Mes to BSO-treated rats resulted in induction of ALA synthetase. m-DCB and 2,4- and 3,5-DCPSO 2Mes produced a dose-related increase in liver levels of methyl sulfones. The changes in the ALA synthetase activity after the administration of varying doses of m-DCB were similar to those after the administration of 2,4- or 3,5-DCPSO 2Mes, whereas the sum of the concentration of two methyl sulfones in the liver of rats dosed with m-DCB was almost the same as the concentration of methyl sulfone after the administration of either 2,4- or 3,5-DCPSO 2Mes. The results strongly suggest that the methyl sulfones derived from m-DCB, i.e., 2,4- and 3,5-DCPSO 2Mes, contribute highly to the induction of the ALA synthetase activity by the parent compound.

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The activities of δ-aminolevulinic acid (ALA) synthetase,1 ALA dehydratase, uroporphyrinogen synthetase, and the quantities of the metabolites, porphobilinogen (PBG), coproporphyrin, and protoporphyrin were serially assayed in the livers of inbred mice which had received repeated injections of 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-trimethylpyridine (DDC). During 48 hr of drug treatment, ALA synthetase activity increased 5- to 20-fold above a basal activity of 30–100 nmoles ALA formed/ hr/g liver. Hepatic uroporphyrinogen synthetase activity did not increase above a basal level of 35–60 nmoles PBG consumed/hr/g liver and appeared to represent the rate-limiting step during most of the course of induction of porphyria. ALA dehydratase activity was dependent upon genotype at the Lv locus and was strain specific, but in all strains this enzyme was present in excess (700–4800 nmoles/hr/g liver). The activity of the dehydratase increased 30% during 48 hr of drug treatment. PBG, proto- and, to a lesser extent, coproporphyrins accumulated, but much less porphyrin was found in livers of C57L/J mice than in those of A/HeJ, C57BL/6J, DBA/2J, SWR/J or C3H/HeJ. Hepatic protoporphyrin accumulation was directly correlated with ALA synthetase activity provided that the protoporphyrin level was less than 50 nmoles/g liver. When hepatic protoporphyrin exceeded 100 nmoles/g liver, there was an inverse correlation between ALA synthetase activity and protoporphyrin content suggesting that either feedback inhibition or feedback repression of the synthetase had occurred.

Role of gastrin/pentagastrin in regulation of intestinal cytochrome P-450

1. 1. In the absence of intraluminal inducers, low “basal” levels of cytochrome P-450 and its dependent MFO activities are detected in the rat intestinal mucosa, and may be regulated by endogenous hormones. 2. 2. Rats were nutritionally maintained by either short term (48 hr) intravenous glucose infusion or chronic (8 days) intravenous hyperalimentation, and were treated with various doses of pentagastrin in the infusate. 3. 3. Regardless of the dose (6–90 μg/kg/hr) or duration of infusion (2–8 days), pentagastrin had no effect on small intestinal cytochrome P-450, its dependent MFO activity, or the activity of δ-aminolevulinic acid synthetase. 4. 4. The intestinal trophic peptide hormone, gastrin, apparently does not regulate the cytochrome P-450-dependent MFO system of the small intestine.

Induction of cytochrome p-450 and related drug-oxidizing activities in muscone (3-methylcyclopentadecanone)-treated rats

In the present study, we investigated the effects of muscone on both in vitro and in vivo parameters of the hepatic microsomal drug-metabolizing enzyme system and other enzyme activities in rats. In the in vivo study, the serum dimethadione (DMO)/trimethadione (TMO) ratios at 2 hr after oral administration of TMO (100 mg/kg) were significantly increased in both male and female rats treated with 75 and 150 but not 40 mg muscone/kg. Antipyrine metabolite profile in 24 hr urine of rats pretreated with muscone (150 mg/kg) was examined. The results showed that the excretion of norantipyrine was significantly increased as compared to the control group. In the in vitro study, we found that the content of cytochrome P-450, and activities of aminopyrine N-demethylase, aniline hydroxylase and δ-aminolevulinic acid (ALA) synthetase were significantly increased as compared to the controls in both male and female rats treated with muscone (75 and 150 mg/kg). This type of induction of the hepatic metabolizing enzymes was similar to that seen after treatment with a prototype drug, phenobarbital.

Effects of phorone (diisopropylidene acetone), a glutathione (GSH) depletor, on hepatic enzymes involved in drug and heme metabolism in rats: Evidence that phorone is a potent inducer of heme oxygenase

Concomitant with the depletion of glutathione content, phorone (250 mg/kg, ip.) produced a marked increase in heme oxygenase activity, biphasic effect on δ-aminolevulinic acid synthetase activity, and slight decreases in cytochrome P-450 content and aminopyrine demethylase activity in the liver of rats. The increase in heme oxygenase activity evoked by phorone was almost completely blocked by pretreatment of rats with actinomycin D and cycloheximide. Phorone was able to produce the changes in these parameters in a dose-dependent manner. Buthionine sulfoximine, a GSH depletor by inhibition of biosynthesis, failed to affect these hepatic parameters.

Aberration of heme and hemoprotein in aged female rats

The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of mono-oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the “regulatory” heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicato of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome- P-450 and its dependent monooxygenase activities in senescent female rats.

Effect of methanol on the activity of hepatic .DELTA.-aminolevulinic acid synthetase in mice.

The effect of methanol on the activity of hepatic δ-aminolevulinic acid synthetase (ALA synthetase) was investigated in male mice. The activity of ALA synthetase increased with increasing doses of methanol up to 100 mmol per kg of body weight injected intraperitoneally. In both the methanol-treated mice and normal mice, the apparent biological half-life time of the enzyme was about 56 min after the administration of cycloheximide. The increase of the enzyme activity by methanol was significantly inhibited by pretreatment with actinomycin D, an inhibitor of protein synthesis. The apparent Km value of the enzyme for glycine did not change but the Vmax value increased in methanol-treated mice. Pretreatment of mice with pyrazole, a potent inhibitor of alcohol dehydrogenase, significantly inhibited the methanol-induced increase of ALA synthetase, whereas pretreatment with either 3-amino-1, 2, 4-triazole or acetanilide, inhibitors of catalase, did not alter the increase of the enzyme activity by methanol. It was concluded that methanol injected to fasted mice induces hepatic ALA synthetase and that the enzyme induction is associated with the oxidation of methanol by alcohol dehydrogenase.