Gamma-glutamyl transferase: A sensitive marker in experimental hexachlorobenzene intoxication

The behaviour of gamma-glutamyl transferase (γ-GT) in rat liver, serum and intestinal mucosa under the effect of hexachlorobenzene (HCB) was studied. The investigation was carried out in order to evaluate γ-GT as a biological parameter in experimental HCB porphyria. HCB was administered by gavage at daily doses of 3.5 mmol/kg body wt for 60 days or 90 days.γ-GT in liver homogenate progressively increased during the treatment. Following a 60-day exposure the enzymatic activity exceeded the values of the controls by more than 60 times. The rise of serum γ-GT was more moderately expressed (5–6-fold).γ-GT in jejunal mucosa was elevated 2-fold after a 90-day treatment. The termination of the daily intoxication reduced back to normal values the enzymatic activity in liver and in intestine as measured after 60 days from the cessation of HCB application.It can be pointed out, that γ-GT is a sensitive and valuable marker for experimental HCB intoxication.     

Early increases in transglutaminase activity and polyamine levels in a Mallory-Denk body mouse model

Rodents treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) are a model of two hepatic toxic manifestations: porphyria and the appearance of hepatic cytoplasmic protein aggregates (Mallory-Denk Bodies, MDBs). MDBs are induced after long-term DDC feeding, consist primarily of keratins 8 and 18, and contain glutamine-lysine cross-links generated by transglutaminases (TGs). TGs are Ca²⁺-dependent enzymes which catalyze the formation of covalent bonds between proteins and between proteins and polyamines. The aim of the current study was to investigate the time-course of TG hepatic activity in CF1 male mice either acutely or chronically treated with DDC and to correlate this activity with polyamine and porphyrin levels. On day 3 of the treatment, statistically significant increases in TG activity (75%), porphyrin content (6740%) and spermidine levels (73%) were observed. Although not statistically significant, at this time point putrescine levels showed an increase of 52%. The highest TG activity was observed on day 30 (522%), while porphyrin levels were still gradually increasing by day 45 (37,000%). From day 7 of the treatment and until the end of the experiment, putrescine levels remained increased (781%). Spermine levels were not affected by the treatment. The DDC-induced increases in putrescine and spermidine levels herein reported seem to be an early event contributing to the stimulation of liver TG activity, and thus to the promotion of cross-linking reactions between keratin proteins. This in turn would contribute to the formation of protein aggregates, which would lead to the appearance of MDBs. Due to the pro-oxidant and antioxidant properties of polyamines, it is possible to speculate that putrescine and spermidine may also participate at several levels in the oxidative stress processes associated with MDB formation.     

Effect of parathion,malathion, endosulfan and chlordane on porphyrin accumulation and alasynthetase in chick embryo liver

In the present study we investigated the porphyrinogenic ability of 4 pesticides: chlordane, endosulfan, parathion and malathion, all of which are widely used in agriculture.In order to determine whether they affect the heme biosynthetic pathway we studied, in 17-day old chick embryo liver “in ovo”, their effects on the amount of porphyrins and on the activity of δ-ALA synthetase (ALA-S), the first and rate limiting enzyme of this pathway.All of them induced hepatic porphyrin accumulation to a different extent compared with dimethylsulfoxide (DMSO) controls. Parathion as well as endosulfan promoted remarkable increases, chlordane raised porphyrin level in a lower degree and malathion slightly modified it. However, the accumulation observed with malathion was markedly enhanced if the period of incubation was extended to 48 h and, even more, if a second dose was injected during these 48 h.When ALA-S activity was analyzed in the chick embryos treated with parathion or endosulfan no alteration could be found in spite of producing noticeable accumulation of porphyrins. In contrast, chlordane promoted a statistically significant elevation of ALA-S as well as malathion which produced the highest induction observed.These results show that not only organochlorinated but also organophosphorous pesticides affect heme metabolism and that induction of ALA-S and porphyrin accumulation are not parallel.     

Reduction in porphyrin excretion as a sensitive indicator of lead toxicity in primary cultures of adult rat hepatocytes

Alterations of specific metabolic pathways can be used as sensitive indicators of toxicity by chemicals and can give valuable information on the mechanism(s) involved. Short-term effects of lead on hepatic haem biosynthesis were studied in an in vitro system. Primary cultures of adult rat hepatocytes were exposed for 24–48 hr to lead (0.024–3.6 m ), and excreted and intracellular porphyrins were measured in untreated and lead-treated cultures. Cytotoxicity, as estimated by enzyme leakage, and morphological alterations were also evaluated. Control hepatocytes produced porphyrins at a rate of 387 pmol/mg cellular protein/day. Most of the released and intracellular porphyrins were protoporphyrins, although uro- and coproporphyrins were also detected in lower amounts. After 24 hr of exposure to 0.1–3.6 m Pb²⁺ , excreted porphyrins decreased by 24–92% and intracellular porphyrins by 36–60%, while 48 hr of exposure to 0.024–3.6 m Pb²⁺ caused a progressive reduction of 77–97% in porphyrin excretion and of 49–67% in intracellular porphyrins. Lead exposure also produced a differential decrease of proto-, copro- and uro-porphyrin excretion. These lead effects can be explained mainly by inhibition of the enzyme 5-aminolaevulinate dehydratase, resulting in a decreased monopyrrole supply for porphyrin biosynthesis, and probably by inhibition of the enzyme uroporphyrinogen decarboxylase. Morphological alterations and enzyme leakage were detected only after 24 hr of exposure to 2.4 m and 48 hr of exposure to 3.6 m Pb²⁺, respectively. The results show that changes in porphyrin production, and particularly in their excretion, in cultured rat hepatocytes are useful indicators of lead toxicity, since they are more sensitive than enzyme leakage and can give preliminary information on the enzyme(s) that could be affected. They also suggest the potential benefits of the use of this method for the evaluation of compounds that alter haem biosynthesis.     

How porphyrinogenic drugs modeling acute porphyria impair the hormonal status that regulates glucose metabolism. Their relevance in the onset of this disease

This work deals with the study of how porphyrinogenic drugs modeling acute porphyrias interfere with the status of carbohydrate-regulating hormones in relation to key glucose enzymes and to porphyria, considering that glucose modulates the development of the disease. Female Wistar rats were treated with 2-allyl-2-isopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) using different doses of AIA (100, 250 and 500 mg/kg body weight) and a single dose of DDC (50 mg DDC/kg body weight). Rats were sacrificed 16 h after AIA/DDC administration. In the group treated with the highest dose of AIA (group H), hepatic 5-aminolevulinic acid synthase (ALA-S) increased more than 300%, phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP) activities were 43% and 46% lower than the controls, respectively, plasmatic insulin levels exceeded normal values by 617%, and plasmatic glucocorticoids (GC) decreased 20%. GC results are related to a decrease in corticosterone (CORT) adrenal production (33%) and a significant reduction in its metabolization by UDP-glucuronosyltransferase (UGT) (62%). Adrenocorticotropic hormone (ACTH) stimulated adrenal production 3-fold and drugs did not alter this process. Thus, porphyria-inducing drugs AIA and DDC dramatically altered the status of hormones that regulate carbohydrate metabolism increasing insulin levels and reducing GC production, metabolization and plasmatic levels. In this acute porphyria model, gluconeogenic and glycogenolytic blockages caused by PEPCK and GP depressed activities, respectively, would be mainly a consequence of the negative regulatory action of insulin on these enzymes. GC could also contribute to PEPCK blockage both because they were depressed by the treatment and because they are positive effectors on PEPCK. These disturbances in carbohydrates and their regulation, through ALA-S de-repression, would enhance the porphyria state promoted by the drugs on heme synthesis and destruction. This might be the mechanism underlying the “glucose effect” observed in hepatic porphyrias. The statistical correlation study performed showed association between all the variables studied and reinforce these conclusions.     

Hexachlorobenzene as hormonal disruptor--studies about glucocorticoids: their hepatic receptors, adrenal synthesis and plasma levels in relation to impaired gluconeogenesis

In Wistar rats, hexachlorobenzene (HCB) depresses the gluconeogenic enzyme phosphoenolpyruvate-carboxykinase (PEPCK). In the liver, glucocorticoids (GC) normally regulate the glucose synthesis by acting on PEPCK. Thus, the aim of this work was to investigate, in a time-course study, the effects of HCB on plasma GC, its adrenal synthesis and stimulation, and the kinetic parameters of its hepatic receptors (GR) in relation to the gluconeogenic blockage produced by HCB. Plasma corticosterone (CORT) concentration, urinary porphyrins and hepatic PEPCK were determined after 2, 4, 6 and 8 weeks of HCB-treatment. The effect of HCB on kinetic parameters of GR was studied in adrenalectomized porphyric rats after 2, 4 and 8 weeks of treatment. Additionally, adrenal CORT synthesis in the same weeks was measured with or without ACTH. Results show that plasma CORT in intoxicated animals dropped significantly after 2 and 4 weeks of treatment (23% and 58%, respectively), and then remained constant until the 8th week. HCB also promoted a reduction in the number of hepatic GR (50-55%) without modifying affinity. After 8 weeks, when porphyria was well established (40-50-fold increase in urinary porphyrins), a reduction (52%) in hepatic GR number, as well as a decrease in PEPCK activity (56%) were observed. Moreover, CORT biosynthesis in adrenals from intoxicated animals significantly decreased (60%) without changes in ACTH effect. Briefly, this paper shows that HCB causes a disruption in GC and GR. This disturbance could contribute to the negative effect on glucose synthesis through PEPCK regulation, thus modulating porphyria. These results enhance the knowledge about the hormonal disruption produced by chlorinated xenobiotics.     

Stimulation of 5-aminolevulinic acid metabolism by uroporphyrinogen I in rat liver homogenate

The effect of excess uroporphyrinogens I and III, coproporphyrinogen III, and the corresponding porphyrins, on the rate of 4-14C-5-aminolevulinic acid (ALA) metabolism was studied. Experiments were performed with mitochondria-free rat liver homogenates prepared from normal rats. The consumption of labelled 5-aminolevulinic acid was followed by measuring its level in aliquots removed at intervals. The pattern of porphyrinogen synthesis was examined by high pressure liquid chromatography. Only uroporphyrinogen I had an effect; it increased the rate of conversion of ALA and porphobilinogen (PBG) to porphyrinogens. Chromatographic analysis revealed increased synthesis of uroporphyrinogen and heptacarboxylic porphyrinogen. It is believed that this mechanism might explain the lack of ALA and PBG accumulation in erythropoietic porphyria and porphyria cutanea tarda, and the absence of acute porphyria attacks in these conditions.     

Hepatic alteration of tryptophan metabolism in an acute porphyria model Its relation with gluconeogenic blockage

This study focuses on the alterations suffered by the serotoninergic and kinurenergic routes of tryptophan (TRP) metabolism in liver, and their relation with gluconeogenic phosphoenolpyruvate-carboxykinase (PEPCK) blockage in experimental acute porphyria. This porphyria was induced in rats by a combined treatment of 2-allyl-2-isopropylacetamide (100, 250, 500 mg/kg bw) and 3,5-dietoxicarbonil 1,4-dihydrocollidine (constant 50 mg/kg bw dose). Results showed a marked dose-dependent increase of all TRP pyrrolase (TRPp) forms, active (holo, total) and inactive (apo), and a decrease in the degree of enzyme saturation by heme. Increases for holo, total, and apo-TRPp were 90, 150, and 230%, respectively, at the highest dose assayed (H). The treatment also impaired the serotoninergic route of TRP metabolism in liver, causing a decrease in serotonin level (H, 38%), and a concomitant enhancement in TRP content (H, 23%). The porphyrinogenic treatment promoted a blockage in PEPCK activity (H, 30%). This occurred in correlation to the development of porphyria, to TRPp alterations and to the production of hepatic microsomal thiobarbituric acid reactive substances. Porphyria was estimated through increases in 5-aminolevulinic acid-synthase (ALA-S) activity, ALA and porphobilinogen contents, and a decrease in ferrochelatase activity. Thus, the TRP kynurenine route was augmented whereas the serotoninergic route was reduced. PEPCK blockage could be partly attributed to quinolinate generated from TRP by the increase of TRPp activity, which would be due to the effect of porphyrinogenic drugs on TRP. The contribution of ROS to PEPCK blockage is analyzed. Likewise, the implication of these results in the control of porphyrias by glucose is discussed.     

Heme and hemoproteins in streptozotocin-diabetic female rats

Alterations in heme biosynthetic and degradative capabilities and in the activities of several heme-containing enzymes were examined in hepatic tissues of streptozotocin (STZ)-diabetic female Sprague-Dawley rats. Activities were measured 10, 30 and 90 days following the administration of STZ (65 mg/kg, i.v.). The activities of the key enzymes involved in heme synthesis, delta-aminolevulinic acid (ALA) synthase, ALA dehydratase, and uroporphyrinogen synthase, were decreased markedly in STZ-diabetic rats as compared to sham-operated animals. Furthermore, the catabolism of heme which occurs via microsomal heme oxygenase (MHO) remained unaltered in these animals. Microsomal content of heme and cytochrome P-450, and the activities of tryptophan pyrrolase and the drug-metabolizing enzymes benzo[a]pyrene (BP) hydroxylase and aniline hydroxylase, were increased in the livers of diabetic rats. By contrast, the activity of the heme-containing enzyme catalase was decreased in these animals. Cobalt chloride produced a marked increase in MHO with a concomitant decrease in microsomal content of cytochrome P-450 and its associated BP hydroxylase activity in normal as well as chronically diabetic rats. It was of interest, however, that the increase in ALA synthase that is normally produced by this metal was not seen in chronic diabetic animals. Thus, chronic diabetes produced subtle and important disruptions in cellular metabolism, which may have been the result of long-term alterations in key enzymes involved in heme synthesis.