An odd case of heteroallelic acute intermittent porphyria in the Argentinean population.

AIP is an acute liver disorder caused by a deficiency of porphobilinogen deaminase (PBGD) characterized by neuroabdominal symptoms. It is an autosomal dominant disease. However, homozygous dominant AIP (HD-AIP) have been described. In some cases erythrodontia was observed. CEP is an autosomal recessive disease produced by mutations in the uroporphyrinogen III synthase gene (UROS), characterized by severe cutaneous lesions and erythrodontia. The aim of the work was to establish the differential diagnosis of porphyria in a patient with abdominal pain, neurological attacks, skin symptoms and erythrodontia. The PBGD activity was reduced 50% and the genetic analysis indicated the presence of two genetic variants in the PBGD gene, p.G111R and p.E258G, a new genetic variant, revealing a case of heteroallelic HD-AIP. The patient, first diagnosed as a carrier of a dual porphyria: AIP / CEP based on the excretion profile of porphyrins, precursors and her clinical symptoms, would be an atypical case of human HD-AIP. These results would also suggest the presence of a phenocopy of the CEP, induced by an endogenous or exogenous factor. Our findings highlight the importance of genetic studies for a proper diagnosis of porphyria, prevention of its manifestation and its treatment.

Possible involvement of stress hormones and hyperglycaemia in chronic mild stress-induced impairment of immune functions in diabetic mice.

Stress, an important aspect of modern life, has long been associated with an altered homeostatic state. Little is known about the effect of the life stress on the outcome of diabetes mellitus, especially related to the higher risk of infections. Here, we evaluate the effects of chronic mild stress (CMS) exposure on the evolution of type I diabetes induced by streptozotocin administration in BALB/c mice. Exposure of diabetic mice to CMS resulted in a significant reduction of survival and a sustained increase in blood glucose values. Concerning the immune response, chronic stress had a differential effect in mice with diabetes with respect to controls, showing a marked decrease in both T- and B-cell proliferation. No correlation was found between splenic catecholamine or circulating corticosterone levels and the proliferative response. However, a significant negative correlation was found between glucose levels and concanavalin A- and lipopolysaccharide-stimulated proliferative responses of T and B cells. A positive correlation between blood glucose and splenic catecholamine concentrations was found in diabetic mice but not in controls subjected to CMS. Hence, the present report shows that diabetic mice show a worse performance in immune function after stress exposure, pointing to the importance of considering life stress as a risk factor for patients with diabetes.

HFE gene mutations in patients with altered iron metabolism in Argentina.

Hereditary Hemochromatosis (HH) is an iron overload syndrome caused by increased duodenal iron absorption, which leads to excessive iron deposition in parenchymal cells of the liver and mayor organs, causing cirrhosis, diabetes, cardiac failure, endocrine complications and arthritis. There are 6 types of HH related to mutations in the genes that encode proteins of iron metabolism. HH Type I is inherited as an autosomal recessive trait of mutations in HFE gene. We investigate the prevalence of C282Y, H63D and S65C mutations in 95 individuals (77 males, 18 females) bearing iron metabolism alterations to establish an early diagnosis of HH. Among this population, 58% carried mutations in the HFE gene (45 males, 10 females). H63D mutation was found in 32.6% of the subjects (29.5% in heterozygocity, 3.15% in homozygocity). S65C mutation was only detected in the heterozygous form (5.3% of the patients), 2 of them carried also H63D mutation. C282Y in heterozygocity was found in 15.8% of the individuals; but only 4.15% carried this mutation in homozygocity. Our findings are in agreement with the prevalence of the Mediterranean origin of most of our patients, where C282Y mutation is not as common as H63D mutation.

Heme oxygenase, aminolevulinate acid synthetase and the antioxidant system in the brain of mice treated with porphyrinogenic drugs.

Several drugs and stress are involved in the triggering of attacks in acute porphyrias. The central nervous system is extremely sensitive to free radical damage because of a relatively low antioxidant capacity. We have demonstrated that mice brain cholinergic system was altered by the effect of some porphyrinogenic agents. The aim of this work was to investigate how known porphyrinogenic drugs affect delta-Aminolevulinic acid synthetase (ALA-S), which is the response of heme oxygenase (HO) to this challenge and to evaluate if the xenobiotics studied develop stress oxidative in mice brain. HO activity was 50-70% induced after chronic Enflurane and Isoflurane anaesthesia, dietary Griseofulvin and starvation. An increase in mRNA HO expression was caused by chronic anaesthesia and Veronal treatments; instead allylisopropilacetamide (AIA) reduced mRNA expression. ALA-S activity was induced by acute administration of anaesthetics (89%), veronal (240%) and ethanol (80%), while ALA-S mRNA expression augmented by chronic administration of enflurane, AIA and veronal. Stress markers such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and malondialdehyde and reduced glutathione levels showed different responses depending on the xenobiotic assayed. In conclusion, some of the drugs studied produced oxidative stress in brain that was confirmed through HO induction and this could be one of the factors leading to porphyric neuropathy.

Interaction of cimetidine with P450 in a mouse model of hepatocarcinogenesis initiation.

Many drugs and xenobiotics are lipophilic and they should be transformed into more polar water soluble compounds to be excreted. Cimetidine inhibits cytochrome P450. The aim of this study was to investigate the preventive and/or reversal action of cimetidine on cytochrome P450 induction and other metabolic alterations provoked by the carcinogen p-dimethylaminoazobenzene. A group of male CF1 mice received a standard laboratory diet and another group was placed on dietary p-dimethylaminoazobenzene (0.5% w w(-1). After 40 days of treatment, animals of both groups received p-dimethylaminoazobenzene and two weekly doses of cimetidine (120 mg kg(-1), i.p.) during a following period of 35 days. Cimetidine prevented and reversed delta-aminolevulinate synthetase induction and cytochrome P450 enhancement provoked by p-dimethylaminoazobenzene. However, cimetidine did not restore haem oxygenase activity decreased by p-dimethylaminoazobenzene. Enhancement in glutathione S-transferase activity provoked by p-dimethylaminoazobenzene, persisted in those animals then treated with cimetidine. This drug did not modify either increased lipid peroxidation or diminution of the natural antioxidant defence system (inferred by catalase activity) induced by p-dimethylaminoazobenzene. In conclusion, although cimetidine treatment partially prevented and reversed cytochrome P450 induction, and alteration on haem metabolism provoked by p-dimethylaminoazobenzene AB, it did not reverse liver damage or lipid peroxidation. These results further support our hypothesis on the necessary existence of a multiple biochemical pathway disturbance for the onset of hepatocarcinogenesis initiation.

On the promoting action of tamoxifen in a model of hepatocarcinogenesis induced by p-dimethylaminoazobenzene in CF1 mice.

BACKGROUND AND AIMS: Tamoxifen (TMX) has proven to be an effective palliative treatment for advanced breast cancer with low reported incidence of side effects. TMX has been demonstrated to be an initiator and/or a promoter in the rat model of hepatocarcinogenesis. To document the long-term effect of TMX in mice treated with p-dimethylaminoazobenzene (DAB), we have investigated the time response action of these drugs on different biochemical parameters. METHODS: A group of animals was placed on dietary DAB (0.5%, w/w) during a period of 28 weeks. Control animals received a standard laboratory diet. Two other groups of non-treated and DAB-treated animals received TMX citrate (0.025%, w/w) in the diet since day 20. RESULTS: The activities of the enzymes involved in heme synthesis and degradation as evaluated in the DAB group was not further affected by TMX. DAB and/or TMX treatment significantly increased the content of total cytochrome P450 and also the activity of glutathione S-transferase indicating liver damage. In all treated groups oxidative stress and an adaptive response of the natural defense system (catalase and superoxide dismutase) were demonstrated. Histological and morphological studies revealed liver cell hyperplasia in DAB treated group; however, only in the DAB+TMX group solid, trabecular and acinar hepatocellular carcinoma was confirmed at the end of the experimental trial. CONCLUSION: We have demonstrated that TMX produced changes in hepatic enzyme activities which may be relevant for the metabolism and disposition of this and/or other drugs. Because liver tumors could be initiated and promoted by several agents which need to be activated, the possible hazard of TMX should be considered. This study reports that long-term treatment with TMX enhances hepatocarcinogenesis induced by DAB. The widespread use of TMX as an anticancer agent adds to the significance of this study.

Preventive aspirin treatment of streptozotocin induced diabetes: blockage of oxidative status and revertion of heme enzymes inhibition.

Some late complications of diabetes are associated with alterations in the structure and function of proteins due to glycation and free radicals generation. Aspirin inhibits protein glycation by acetylation of free amino groups. In the diabetic status, it was demonstrated that several enzymes of heme pathway were diminished. The aim of this work has been to investigate the in vivo effect of short and long term treatment with acetylsalicylic acid in streptozotocin induced diabetic mice. In both treatments, the acetylsalicylic acid prevented delta-aminolevulinic dehydratase and porphobilinogen deaminase inactivation in diabetic mice and blocked the accumulation of lipoperoxidative aldehydes. Catalase activity was significantly augmented in diabetic mice and the long term treatment with aspirin partially reverted it. We propose that oxidative stress might play an important role in streptozotocin induced diabetes. Our results suggest that aspirin can prevent some of the late complications of diabetes, lowering glucose concentration and probably inhibiting glycation by acetylation of protein amino groups.

Drug metabolizing enzyme system and heme pathway in hepatocarcinogenesis.

Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre-initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2-allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis.

beta-Carotene partially prevents the damage induced by 1,4-dimethylaminoazobenzene in mice.

Hepatocarcinogenesis (HC) induced by various carcinogens such as 1,4-dimethylaminoazobenzene (DAB) is a multistep and complex process. The anticancer efficacy of beta-carotene (beta C) was evaluated by estimating some biochemical parameters during the initiation stage of HC. beta C dietary supplementation partially prevented the rise in delta-aminolevulinate synthetase activity. P 450 levels were dramatically enhanced in all groups studied. beta C administration did not overcome catalase inactivation due to DAB treatment; however, superoxide dismutase activity levels showed to be less decreased in the DAB + beta C animals in comparison to the DAB group. The great enhancement provoked by DAB of glutathione S-transferase, a proposed marker of HC, was partially reversed by beta C. In conclusion, heme pathway regulation, drug metabolism, and natural oxidative defence systems, strikingly modified in DAB fed animals, were partially controlled by provitamin A. The potential use of beta C in preventing carcinogenesis is suggested.

Reducing sugars trigger delta-aminolevulinic dehydratase inactivation: evidence of in vitro aspirin prevention.

1. The effect of in vitro glycation on delta-aminolevulinic dehydratase (ALA-D) under several experimental conditions was investigated. When preincubated with 500 mM glucose at 37 degrees C for 20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold. 2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation cannot be attributed to glucose autoxidation. 3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose. 4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of red blood cells, which can also be applied to sugars other than glucose.

Hepatic enzymatic metabolism alterations and oxidative stress during the onset of carcinogenesis: protective role of alpha-tocopherol.

Oxidants play a role in several stages of carcinogenesis. A high antioxidant capacity is expected to protect 'initiated' cells from excessive oxidant toxicity. The aim of this study was to determine the chemopreventive effect of a-tocopherol (alpha-T) on the hepatocarcinogenesis induced with p-dimethylaminoazobenzene (DAB) in mice. The dietary administration of alpha-T completely reversed the induction of delta-aminolevulinate synthetase and glutathione-S-transferase (the tumoral marker enzyme). alpha-T greatly enhanced P 450 levels, which were even higher in animals exposed to DAB. Indirect evidence for the involvement of oxygen radicals in the DAB model of hepatocarcinogenesis was provided by increased levels of thiobarbituric acid reactive species, which were detected in animals with severe liver damage and were assessed by histological analysis. alpha-T reduced the degree of hepatic injury, although this vitamin produced only slight changes in the oxidative parameters evaluated. The use of alpha-T as a potential chemopreventive agent, particularly during the initiation stage of carcinogenesis provoked by DAB, is worthy of further study.

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 delta-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 alpha-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 alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.

[Changes in the heme metabolic pathway in diabetic patients]. / Alteraciones en el camino metabólico del hemo en pacientes diabéticos.

In the last decades several authors have observed a frequent association between diabetes mellitus and porphyria, mainly porphyria cutanea tarda. In previous studies, it has been demonstrated that both d delta d-aminolevulic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), enzymes of the heme pathway, are inhibited by high concentrations of glucose in vitro in crude preparations of erythrocytes. The activity of these same enzymes was diminished in different tissues obtained from streptozotocin induced diabetic mice. Therefore, we decided to investigate the incidence of heme metabolism alterations in diabetes mellitus in a population of 100 non selected adult patients. The activities of erythrocytic ALA-D and PBG-D were measured. Rhodanese, an enzyme of the sulfocompounds pathway closely related to the regulation of heme biosynthesis, was also studied. Urine porphyrin content as well as the chromatographic pattern of esterified porphyrins were determined. ALA-D and PBG-D activities were diminished in diabetic patients (40% and 20% respectively), while rhodanese was only slightly increased (Fig. 1). ALA-D activity was subnormal in a 92% of the complete diabetic population, while PBG-D activity was less than normal in a 79% of the same population. No significative differences between enzymic activities were observed in the groups insulin and non-insulin dependent (Fig. 3). Urine porphyrin content was increased in 5% of the diabetic population. Chromatographic pattern of urinary porphyrins was notably altered in diabetic patients irrespectively of their porphyrin content (Fig. 4), suggesting an alteration in the enzyme uroporphyrinogen decarboxylase resembling the primary enzymic defect observed in porphyria cutanea tarda.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteraciones en el camino metabólico del hemo en pacientes diabéticos. / [Changes in the heme metabolic pathway in diabetic patients]

In the last decades several authors have observed a frequent association between diabetes mellitus and porphyria, mainly porphyria cutanea tarda. In previous studies, it has been demonstrated that both d delta d-aminolevulic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), enzymes of the heme pathway, are inhibited by high concentrations of glucose in vitro in crude preparations of erythrocytes. The activity of these same enzymes was diminished in different tissues obtained from streptozotocin induced diabetic mice. Therefore, we decided to investigate the incidence of heme metabolism alterations in diabetes mellitus in a population of 100 non selected adult patients. The activities of erythrocytic ALA-D and PBG-D were measured. Rhodanese, an enzyme of the sulfocompounds pathway closely related to the regulation of heme biosynthesis, was also studied. Urine porphyrin content as well as the chromatographic pattern of esterified porphyrins were determined. ALA-D and PBG-D activities were diminished in diabetic patients (40

and 20

respectively), while rhodanese was only slightly increased (Fig. 1). ALA-D activity was subnormal in a 92

of the complete diabetic population, while PBG-D activity was less than normal in a 79

of the same population. No significative differences between enzymic activities were observed in the groups insulin and non-insulin dependent (Fig. 3). Urine porphyrin content was increased in 5

of the diabetic population. Chromatographic pattern of urinary porphyrins was notably altered in diabetic patients irrespectively of their porphyrin content (Fig. 4), suggesting an alteration in the enzyme uroporphyrinogen decarboxylase resembling the primary enzymic defect observed in porphyria cutanea tarda.(ABSTRACT TRUNCATED AT 250 WORDS)

Heme biosynthesis pathway regulation in a model of hepatocarcinogenesis pre-initiation.

1. Heme regulation before the appearance of hyperplastic nodules was investigated in mice models of hepatocarcinogenesis. 2. With this aim 5-aminolaevulinate synthetase (ALA-S), microsomal heme-oxygenase (MHO), mitochondrial and cytoplasmic rhodanese activities were examined throughout a period of 35 days in animals exposed to dietary p-dimethylaminoazobenzene (DAB). 3. ALA-S activity was significantly diminished (50%) on day 14, then showing a sharply rising profile from day 28 onwards, and reaching 350% on day 35. 4. A similar profile was observed for mitochondrial rhodanese activity. 5. Changes in MHO and cytoplasmic rhodanese activities were almost the opposite to those observed for ALA-S. 6. The distinctive alteration in mitochondrial and cytoplasmic rhodanese would suggest that it plays a subtle role in ALA-S regulation during carcinogenesis initiation through a mechanism that appears to involve subcellular localization controls perhaps by means of the breakage of cystine trisulphide postulated to act as an ALA-S activator. 7. Taking into account the present results, we suggest a probable mechanism for the onset of hepatocarcinogenesis that includes a primary activating liver status, provoking biochemical aberration leading to the stage of initiation of hepatocarcinogenesis involving the whole organ.