Atorvastatin shows antitumor effect in hepatocellular carcinoma development by inhibiting angiogenesis via TGF-ß1/pERK signaling pathway.

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins may reduce HCC incidence. Its antitumor activities may be mediated by disrupting several hepatocarcinogenic pathways. To evaluate in vivo and in vitro the antiproliferative and antiangiogenic action of atorvastatin (AT) in the development of HCC as well as its mechanisms of action. In vivo model: hexachlorobenzene (HCB) was used to promote the development of HCC in Balb/C nude mice. Number of hepatic tumor, liver cell proliferation parameters (proliferating cell nuclear antigen, PCNA), angiogenesis, and VEGF levels were analyzed. In vitro model: Hep-G2 and Ea-hy926 cells were used to evaluate the effect of different doses of AT on HCB induced cell proliferation, migration, and vasculogenesis and to analyze proliferative parameters. In vivo: AT prevented liver growth and tumor development and inhibited PCNA, TGF-ß1, and pERK levels increase. AT prevented skin blood vessel formation. In vitro, AT prevented cell proliferation and migration as well as tubular formation in the endothelial cell line by inhibiting the MAPK ERK pathway. We were able to demonstrate the potential AT antiproliferative and antiangiogenic effects in an HCC model and the involvement of TGF-ß1 and pERK pathways.

Abnormal kinetic behavior of uroporphyrinogen decarboxylase obtained from rats with hexachlorobenzene-induced porphyria.

Uroporphyrinogen decarboxylase is an essential enzyme in all organisms and functions in the heme biosynthetic pathway, catalyzing the decarboxylation of the four acetate groups of uroporphyrinogen to form coproporphyrinogen. This work examines whether the four sequential decarboxylations occur at the same active site, and explores whether hexachlorobenzene-induced porphyria affects the behavior of the enzyme. For this purpose, kinetic competition studies were done with mixtures of uroporphyrinogen III and pentacarboxyporphyrinogen III. With the enzyme from normal rats, a constant velocity was obtained with all the mixtures, indicating that uroporphyrinogen and pentacarboxy-porphyrinogen react at the same active site, i.e. the first and fourth decarboxylations occur at the same site. In contrast, in experiments with enzyme from rats with hexachlorobenzene-induced porphyria, the total rate for mixtures was always lower than the reference rate; and a curve with a deep minimum was obtained, indicating that the two reactions occur at functionally different sites, but with cross-inhibition. This suggests that the modifications induced in the enzyme by hexachlorobenzene cause the two active sites to become nonequivalent and functionally different. The question is discussed how the hexachlorobenzene treatment may produce this abnormal kinetic behavior, and alternative hypotheses are considered.

Tryptophan metabolism via serotonin in rats with hexachlorobenzene experimental porphyria.

One of the three pathways for the metabolisation of dietary tryptophan is the formation of serotonin. Tryptophan hydroxylase catalyses the formation of 5-hydroxytryptophan, the first and regulatory step of this biosynthesis. The aim of the present work is to study alterations in this tryptophan metabolism in rats with experimental Porphyria Cutanea Tarda induced by hexachlorobenzene. With this purpose, the content of tryptophan and its metabolites related to the serotonin pathway are determined by HPLC techniques, in tissues (brain, liver and gut) and in fluids (blood, plasma and urine) of controls and hexachlorobenzene-porphyric rats. In these experimental-porphyric animals, we determine a significant increase in the excretion of 5-hydroxyindole acetic acid in urine and a decrease in the content of serotonin in small gut, respect to controls. Significant increases in contents of serotonin in 24-hr urine and tryptophan in liver are also found. No other significant variations for the different metabolites are detected in any of the tissues and fluids studied. Brain and liver activities of the rate-limiting enzyme tryptophan hydroxylase can only be measured in porphyric rats. Our results agree with an increased turnover of gastrointestinal serotonin derived from dietary tryptophan and its excretion as urinary 5-hydroxyindole acetic acid, which is formed in liver. An increased serotonin pathway in porphyric livers is confirmed by the measured increase in the activity of hepatic tryptophan hydroxylase. The absence of neurological symptoms in patients with Porphyria Cutanea Tarda could be related to the absence of a statistically significant variation in serotonin content shown in brain.

Effects of the porphyrinogenic compounds hexachlorobenzene and 3,5-diethoxycarbonyl-1,4-dihydrocollidine on polyamine metabolism.

The naturally occurring polyamines--putrescine, spermidine and spermine--are organic cations present in all living cells and essential for cell growth and differentiation. The aim of the present study was to extend the investigations on the effects of porphyrinogenic compounds on polyamine metabolism. This was achieved by studying putrescine, spermidine and spermine levels in a model of acute porphyria, i.e. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced porphyria, and in a model of non-acute porphyria, i.e. hexachlorobenzene (HCB)-induced porphyria. HCB administration to female Wistar rats for 7, 14, 21, 28 and 56 days did not alter polyamine levels in liver, even though rats presented clear signs of HCB-induced porphyria. In contrast to HCB, DDC treatment resulted in a remarkable increase in putrescine levels in the liver of female and male Sprague-Dawley rats. This increase was due, at least in part, to ornithine decarboxylase (ODC) activation. DDC induction of putrescine levels did not show organ specificity, since it could also be seen in adrenal gland. Interestingly, the deregulation of polyamine biosynthesis occurred concomitantly with the deregulation of the heme biosynthetic pathway. In addition to porphyria, it is known that DDC intoxication affects several proteins of the hepatocyte cytoskeleton. It is suggested that DDC-induced increase in ODC activity and putrescine levels may be an early event contributing to alter the cytoskeleton.

Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyria-independent.

Hexachlorobenzene (HCB) alters phospholipid and heme metabolisms in the liver and Harderian gland. The effects of HCB on phospholipid metabolism, in an organ considered to be non-responsive to its porphyrinogenic effects, remain to be studied. Therefore, as the brain is an organ with this feature, this paper analyzes the effects of HCB on brain phospholipid composition in order to investigate if there is any relationship between HCB-induced porphyrin metabolism disruption and phospholipid alterations. For this purpose, a time-course study of HCB effects on brain phospholipids was performed in two strains of rats differing in their susceptibility to acquire hepatic porphyria: Chbb THOM (low); and Wistar (high). This paper shows for the first time that rat brain phospholipids are affected by HCB exposure. Comparative studies show that HCB-induced disturbances in brain phospholipid patterns are time and strain-dependent. Thus, whereas major phospholipids, phosphatidylcholine and phosphatidylethanolamine were more altered in Wistar rats, minor phospholipids, phosphatidylinositol and phosphatidylserine were more affected in Chbb THOM rats. HCB intoxication led to a sphingomyelin/phosphatidylcholine molar ratio lower than the normal, in both strains. As was expected, brain porphyrin content was not altered by HCB intoxication in either strain. It can be concluded that HCB is able to alter brain phospholipid metabolism in a strain-dependent fashion, and in the absence of alterations in brain heme metabolism. In addition, HCB-induced disturbances in brain phospholipids were not related to the degree of hepatic porphyria achieved by the rats.

How does hexachlorobenzene treatment affect liver uroporphyrinogen decarboxylase?

The aims of the present work were: (1) to investigate whether the strong decrease of liver uroporphyrinogen decarboxylase (UroD) activity observed in experimental porphyria cutanea tarda is due to alteration of the enzymatic protein and (2) to improve the knowledge about the normal liver enzyme. With these purposes, several physicochemical studies for enzymatic characterization were carried out comparatively on the 12-fold purified liver enzyme of both normal and hexachlorobenzene porphyric rat. The study shows that the enzyme from porphyric rats has a higher activation energy, lower reactivity index and lower optimum pH than the normal one. In addition, it did not reach the Vmax at any of the substrate concentrations assayed (up to 28 microM uroporphyrinogen III), while the normal enzyme reached the plateau around 14 microM. The porphyric enzyme appears to be more protected than the normal against the inhibitory action of several metals, particularly Cu2+ and Pb2+, and against thermal inactivation. Zn2+ did not affect enzymatic activity, whereas Cu2+, Hg2+, Fe2+, Pb2+, and Cd2+ lowered the activities of both normal and porphyric enzyme in a dose-related way. It was also observed that the larger the atomic radius in its hydrated state, the lower the effect of the metal. Neither glutathione nor dithiothreitol significantly altered enzymatic activity in the range of concentrations assayed. beta-Mercaptoethanol had diverse effects, as regards both the concentration assayed and the enzymatic sample used. Assays with cystine showed a dual behaviour of both normal and porphyric enzymatic activity. Western blots for both preparations revealed a single band (65 kDa) with a similar intensity. This study show that hexachlorobenzene treatment modifies the physicochemical properties of liver UroD leading to a sharp decrease of its activity, without affecting its antigenic reactivity probably as a consequence of changes at the conformational level promoted by the binding of its reported inhibitor.

Effect of hexachlorobenzene on NADPH-generating lipogenic enzymes and L-glycerol-3-phosphate dehydrogenase in brown adipose tissue.

The effect of the in vivo administration of hexachlorobenzene (HCB) (100 mg/100 g bw) for 30 days, on the activities of brown adipose tissue (BAT) lipogenic enzymes, i.e. malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PD) and the mitochondrial non lipogenic enzyme, L-glycerol-3-phosphate dehydrogenase (LG3PD), was studied in male Wistar rats, submitted to various neurohormonal manipulations. BAT ME, G6PD and LG3PD activities showed significant reductions in response to HCB treatment both in euthyroid and surgically thyroidectomized rats, showing that the effect does not depend on the presence of thyroid hormones. These results differ from those obtained for hepatic ME and G6PD activities, which increased in HCB intoxicated rats without alteration in LG3PD. HCB decreased BAT ME activity under BAT denervation. Administration of HCB resulted in time and dose-dependent decreases in the activity of BAT malic enzyme. The basal activity of ME was increased in hypothyroid rats, while that of LG3PD was reduced. A stimulatory effect of receptor-saturating doses of triiodothyronine (T3) (50 microg/100 g body weight) was observed on BAT ME and LG3PD activities in thyroidectomized rats, showing that the enzymes responded to thyroid hormone stimulation in a normal manner. The stimulatory effect of saturating doses of T3 on ME and LG3PD was reduced by HCB. The results presented herein unequivocally show that brown adipose tissue is a specific target in HCB-induced toxicity, which in turn involves severe alterations in the regulation of BAT lipogenesis.

Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme.

Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.

Hexachlorobenzene-induced alterations of rat hepatic microsomal membrane function.

The time and dose-dependent effects of the in vivo administration of hexachlorobenzene (HCB), on hepatic microsomal membrane functions, were studied in female Wistar rats. Administration of HCB (100 mg/100 g b.w.) resulted in time-dependent decreases in the activity of two membrane-bound enzymes: 5'nucleotidase and Na+/K+ ATPase. HCB was found to cause a significant rise in protein tyrosine kinase (PTK) activity during the early stages of intoxication (day 2), followed by a significant decrease at 10 days, returning to control levels after 20 days of treatment. A stimulatory effect of HCB on in vitro endogenous microsomal protein phosphorylation was observed from 2 days of intoxication up to 30 days of treatment, with an important stimulation of phosphorylation at 5 days. Administration of HCB (100 mg/100 g b.w.) for 10 days caused a 50% reduction in epidermal growth factor receptor (EGF-R) ligand binding. The effects of known specific inhibitors of protein phosphatases on endogenous protein phosphorylation were studied. HCB affected the labelling of several bands, as well as the 5'nucleotidase and PTK activities, in a dose-dependent manner. In conclusion, this study indicated that the in vivo administration of HCB results in a significant alteration of membrane function.

Precancerous pathology evoked by hexachlorobenzene treatment.

The porphyrinogenic and carcinogenic ability of hexachlorobenzene (HCB) was assayed in male and female gold hamsters, and histological examinations of tissue alterations were performed. So it was studied, in liver: a) porphyrin content which was significantly increased at five months of HCB treatment, specially in males, and the pattern of accumulated porphyrins which was altered independent of the sex, b) haem pathway enzymes:delta aminolaevulinic acid synthase, ferrochelatase and porphyrinogen carboxylyase (PCL); among which only PCL appeared to be altered just at ten months of HCB feeding. While thyroid gland and kidney remained unaltered along the treatment time, liver and spleen exhibited a noticeable size variation and morphological alterations. In fact the spleen in treated animals was hypotrophic showing a red pulp less developed with respect to the Malpighian corpuscles and many macrophages with iron deposits. Respect to the liver, enlargement in size of hepatocytes, high content of iron deposits, no PAS positive structures in the cytoplasm, several small lipid droplets, microsteatosis although no cytonecrosis, polymorphic nuclei, and proliferations of nucleoli were observed. Therefore HCB is able to cause precancerous pathology and to induce porphyria in hamsters, but not hyperthyroidism, upon this experimental conditions. By the way, males were found to be a good experimental model, better than females, to study the earliest relations between porphyria and cancer.

Precancerous pathology evoked by hexachlorobenzene treatment

The porphyrinogenic and carcinogenic ability of hexachlorobenzene (HCB) was assayed in male and female gold hamsters, and histological examinations of tissue alteraions were performed. So it was studied, in liver: a) prophyrin content which was significantly increased at five months of HCB treatment, specially in males, and the pattern of accumalated porphyrins which was altered independent of the sex, b) haem pathway enzymes: delta aminolaevulinicacid synthase, ferrochelatase and porphyrinogen carboxylyase (PCL); among which only PCL appeared to be altered just at ten months of HCB feeding. While thyroid gland and kidney remained unaltered along the treatment time, liver and spleen exhibited a noticeable size variation and morphological alterations. In fact the spleen in treated animals was hypotrophic showing a red pulp less developed with respect to the Malpighian corpuscles and many macrophages with iron deposits. Respect to the liver, enlargement in size of hepatocytes, high content of iron deposits, no PAS positive structures in the cytoplasm, several small lipid droplets, microsteatosis although no cytonecrosis, polymorphic nuclei, and proliferations of nucleoli were observed. Therefore HCB is able to cause precancerous pathology and to induce porphyria in hamster, but not hyperthyroidism, upon this experimental conditions. By the way, males were found to be a good experimental model, better than females, to study the earliest relations between porphyria and cancer. (AU)

Precancerous pathology evoked by hexachlorobenzene treatment

The porphyrinogenic and carcinogenic ability of hexachlorobenzene (HCB) was assayed in male and female gold hamsters, and histological examinations of tissue alteraions were performed. So it was studied, in liver: a) prophyrin content which was significantly increased at five months of HCB treatment, specially in males, and the pattern of accumalated porphyrins which was altered independent of the sex, b) haem pathway enzymes: delta aminolaevulinicacid synthase, ferrochelatase and porphyrinogen carboxylyase (PCL); among which only PCL appeared to be altered just at ten months of HCB feeding. While thyroid gland and kidney remained unaltered along the treatment time, liver and spleen exhibited a noticeable size variation and morphological alterations. In fact the spleen in treated animals was hypotrophic showing a red pulp less developed with respect to the Malpighian corpuscles and many macrophages with iron deposits. Respect to the liver, enlargement in size of hepatocytes, high content of iron deposits, no PAS positive structures in the cytoplasm, several small lipid droplets, microsteatosis although no cytonecrosis, polymorphic nuclei, and proliferations of nucleoli were observed. Therefore HCB is able to cause precancerous pathology and to induce porphyria in hamster, but not hyperthyroidism, upon this experimental conditions. By the way, males were found to be a good experimental model, better than females, to study the earliest relations between porphyria and cancer.

Hepatic indices of thyroid status in rats treated with hexachlorobenzene.

The functional thyroid status of hexachlorobenzene (HCB)-treated rats was studied. HCB caused a depletion of serum thyroxine (T4), but did not change L-3,5,3'-triiodothyronine (T3) levels in serum of rats. The activities of the thyroid regulated mitochondrial enzyme L-glycerolphosphate dehydrogenase (LGPD) and cytosolic enzymes, malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were assayed in livers of normal and HCB (100 mg/100 g bw) treated Wistar rats. Mitochondrial LGPD activity did not change significantly, however ME, 6GPD and G6PD were induced by HCB only in non-thyroidectomized animals. The absence of cytosolic enzymes induction in thyroidectomized rats treated with HCB indicates that HCB is not intrinsically thyromimetic. The induction of hepatic ME, G6PD and 6PGD activities in HCB thyroidectomized rats was dependent on the presence of thyroid hormone. The unchanged activity of mitochondrial LGPD in contrast to the increased activities of the cytosolic enzymes ME, G6PD and 6PGD is not consistent with a shift in functional thyroid status following HCB treatment.

Influence of hepatic tumors caused by diethylnitrosamine on hexachlorobenzene-induced porphyria in rats.

The response of female BDVI rats bearing diethylnitrosamine(DENA)-induced hepatic tumors to the porphyrinogenic action of hexachlorobenzene (HCB) was studied. (1) The heme pathway operates in these tumors but they were less affected by HCB than the liver. (2) Tumors did not accumulate porphyrins although the surrounding liver accumulated more porphyrins than livers treated with HCB. (3) DENA/HCB livers which developed a well defined tumor showed slightly less porphyrinogen carboxylyase inhibition and delta-aminolaevulinate synthase induction than HCB rats. (4) The results of the present work suggest that endogenously formed porphyrins would be unable to be accumulated by DENA-induced tumors when the tumoral development precedes the onset of the porphyria.

Liver ferrochelatase from normal and hexachlorobenzene porphyric rats. Mechanism of drug action.

1. The action of hexachlorobenzene (HCB) on hepatic ferrochelatase was investigated. 2. A direct action of HCB, pentachlorophenol, porphyrins and haem on this enzyme activity was discarded. 3. In HCB porphyric liver there is probably an activator tightly bound to the enzyme. 4. Pyridoxal phosphate (PPL) may be a cofactor of ferrochelatase from both normal and porphyric rats. 5. The PPL would be involved in the binding site of Fe2+ or at least in the approaching of Fe2+ to the active site of the enzyme. 6. The differences found between normal and porphyric preparations could be attributed to conformational changes elicited by the HCB.

Studies on the active centre of rat liver porphyrinogen carboxylase in vivo effect of hexachlorobenzene.

1. Porphyrinogen carboxylase from the liver of normal and hexachlorobenzene porphyric rats was subjected to chemical modification using photo-oxidation with methylene blue, diethylpyrocarbonate, butane-2,3-dione, and phenylglyoxal. 2. All of these chemicals inactivated the enzyme from both sources. 3. Reversion of the diethylpyrocarbonate reaction with hydroxylamine as well as protection of the enzymes with uroporphyrinogen III indicated that histidine is involved at least in the first decarboxylation active site of the porphyrinogen carboxylyase, and perhaps in one or more sites where the removal of the other carboxyl groups take place. 4. Arginine seems not to be at the active site of the enzyme but at its environment since two diketones alter the enzyme activity, however the substrate did not protect the enzyme from the butane-2,3-dione modification. 5. Comparative studies between the enzyme from normal and porphyric animals suggest that the low enzyme activity from intoxicated animals could be due to alterations of its active centre environment produced by hexachlorobenzene treatment. This treatment seems to partially protect the active site of the porphyrinogen carboxylase from the modification reactions.

Liver ferrochelatase from normal and hexachlorobenzene porphyric rats. Studies on their properties.

1. The aim of the present work is to shed light on the way of action of hexachlorobenzene (HCB) on hepatic ferrochelatase the mitochondrial enzyme which catalyzes the last step of haem biosynthetic pathway. 2. Some properties of this enzyme from normal and HCB porphyric rat liver were studied. 3. The present findings indicate that HCB treatment would modify the configuration of the enzyme perhaps allowing the active center of the porphyric ferrochelatase to be more exposed. 4. As a consequence it would show: (a) its higher affinity for the iron; (b) the shorter time necessary to form the intermediate enzyme-substrate, reflected both by the existence of a shorter lag and consequently a shorter pre incubation time. 5. However this modification elicited by the fungicide does not alter the submitochondrial distribution of the enzyme nor the optimal conditions for its measurement.

The role of iron in the hexachlorobenzine induced porphyria: I. Studies on diferent types of iron and its relation with porphyrinogen carboxy-lyase decrease

Se llevaron a cabo estudios para elucidar si en la porfiria por hexaclorobenceno (HCB) están alterados los contenidos de hierro total, no hémico en hígado y si existe alguna relación entre estas alteraciones y el decrenento de la porfirinógeno carboxi-liasa (PCL) hepática en ratas tratadas con la droga. Se observó que en los hígados porfíricos los niveles de hierro total y no hémico incrementaron significativamente como consecuencia de la intoxicación por HCB, mientras que este tratamiento produjo un decremento no significativo en el contenido de hierro hémico. Preparaciones enzimáticas de hígados porfíricos filtradas a través de columnas de Sephadex G-25, que separan el hierro libre y que tienen un contenido de hierro-proteína mayor que las normales, exhibieron una fuerte inhibición de PCL. Agentes quelantes, alfa alfa' bipiridilo y 8-hidroxiquinolina, no revierten tal inhibición. Se ensayó también el efecto in vitro de diferentes concentraciones de hierro inogánico, ferritina y hemina sobre la actividad de PCL normal. Así se observó que hierro inogánico y hemina producen ligera inhibición de PCL cuando se añaden en concentraciones mayores que las correspondientes a un hígado porfírico (0.08 mM y 10-6 M respectivamente como promedio en el medio de incubación). Por lo tanto dichas inhibiciones no tienen significado fisiológico. La ferritina no modifica el proceso de descarboxilación. De estos resultados surge que el hierro no desempeña un papel directo en el decremento de la actividad PCL en la porfiria experimental por HCB, no siendo el inhibidor que se pone de manifiesto en los ensayos de calentamiento. El hierro podría quizás estimular la metabolización del HCB dando origen a metabolitos activos