Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride

In acute liver injury induced by the injection of CCl4, cell death has been attributed to the necrosis of hepatocytes in the centrilobular area. In the present study, we re-examined the hepatic injury evoked by CCl4 in rats and explored the possibility that apoptosis may also contribute to its pathogenesis. Apoptotic hepatocytes were identified and quantified by light and electron microscopy, the in situ immunohistochemical labeling of nuclear DNA fragmentation, flow cytometry, and DNA gel electrophoresis. We found that a substantial number of hepatocytes underwent apoptosis. Apoptotic changes were also observed in ballooned hepatocytes. Apoptotic hepatocytes increased in number at 3 hours and peaked at 6 hours after the CCl4 injection. Apoptotic bodies were sequestrated in the adjacent hepatocytes and sinusoidal cells. Double staining of the cells with immunostaining for phagocytes and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining for labeling of DNA fragmentation showed that the majority of apoptotic hepatocytes were phagocytosed by Kupffer cells and macrophages. The results indicated that apoptosis occurs in the ballooned and injured hepatocytes of the centrilobular area. What occurs after CCl4 administration may be important in reducing inflammation, shortening the course of acute hepatic injury, and preventing the development of fibrosis.     

Evaluation of sex difference in tissue repair following acute carbon tetrachloride toxicity in male and female Sprague-Dawley rats

The formation of zinc protoporphyrin in response to lead or iron depletion has previously been investigated in erythroid systems. Because of its possible metabolic role in non-erythroid tissue, we investigated the formation of zinc protoporphyrin in cultured hepatocytes. The effects of lead and inhibitors of ferrochelatase, the iron insertion step of heme synthesis, on the conversion of 5-aminolevulinic acid to zinc protoporphyrin, protoporphyrin and heme were compared in rat and chick embryo hepatocyte cultures. In rat cultures, zinc protoporphyrin was synthesized enzymatically by ferrochelatase, since N-methylmesoporphyrin, an inhibitor of ferrochelatase. caused 40% or greater decreases in both heme and zinc protoporphyrin accumulation and markedly stimulated protoporphyrin accumulation. In addition, chelation of ferrous iron with 2,2'-dipyridyl decreased heme accumulation by 50%, but increased ZPP accumulation by 200%. Zinc protoporphyrin formation in chick embryo hepatocytes required the addition of zinc as well as 5-aminolevulinic acid and apparently was non-enzymatic, since it was not inhibited by N-methylmesoporphyrin nor increased by iron chelation. In the presence of 5-aminolevulinic acid, lead had no effect on zinc protoporphyrin, protoporphyrin or heme accumulation in chick hepatocytes, but decreased all three in rat hepatocytes, with the decrease in protoporphyrin being far greater than that of zinc protoporphyrin or heme. These findings indicate that, in contrast to the effect of lead in erythroid tissue, it did not specifically increase zinc protoporphyrin accumulation or alter iron availability in cultured hepatocytes.     

Mitochondrial respiratory enzyme function and superoxide dismutase activity following brain glutathione depletion in the rat

In substantia nigra from patients with Parkinson's disease, there are decreased levels of reduced glutathione (GSH) and diminished activities of mitochondrial complex I and alpha-ketoglutarate dehydrogenase (alpha-KGDH), along with increased activity of superoxide dismutase (SOD). However, the interrelationship among these events is uncertain. We now report the effect of decreased brain GSH levels on SOD and mitochondrial respiratory enzyme activity in rat brain. In addition, we have investigated the ability of thioctic acid, an endogenous antioxidant, to alter these parameters. Unilateral or bilateral intracerebroventricular (ICV) administration of buthionine sulphoximine (BSO; 1 x 3.2 mg or 2 x 1.6 mg) over a 48-hr period reduced cortical GSH by 55-70%. There was no change in the activity of complex I, II/III, or IV or of citrate synthase in cortex. Similarly, there was no alteration of mitochondrial or cytosolic SOD activity. Thioctic acid (50 or 100 mg/kg IP) alone had no effect on cortical GSH levels in control animals and did not reverse the decrease in GSH levels produced by unilateral or bilateral ICV BSO administration. Thioctic acid (50 or 100 mg/kg IP) had no overall effect on complex I, II/III, or IV or on citrate synthase activity in control animals. Thioctic acid also did not alter cortical mitochondrial respiratory enzyme activity in BSO-treated rats. At the lower dose, thioctic acid tended to increase mitochondrial and cytosolic SOD activity in control animals and in BSO-treated rats. However, at the higher dose, thioctic acid tended to decrease mitochondrial SOD activity. Overall, there was no consistent effect of thioctic acid (50 or 100 mg/kg IP) on SOD activity in control or BSO-treated animals. This study shows that BSO-induced glutathione deficiency does not lead to alterations in mitochondrial respiratory enzyme activity or to changes in SOD activity. GSH depletion in Parkinson's disease therefore may not account for the alterations occurring in complex I and mitochondrial SOD in substantia nigra. Thioctic acid did not alter brain GSH levels or mitochondrial function. Interestingly, however, it did produce some alterations in SOD activity, which may reflect either its antioxidant activity or its ability to act as a thiol-disulphide redox couple.     

The effect of emotional-painful stress, hypoxia, and adaptation to it on the activity of enzymes for metabolizing glutathione and concentration of glutathione in rat organs

The stress activates glutathione peroxidase in the heart, liver, and kidney, glutathione transferase in the heart and liver, inhibits gamma-glutamyl transferase in the liver; the activity of glutathione reductase and the content of reduced glutathione were unchanged. Two-four-minute hypercapnic hypoxia unchanged the activity of glutathione metabolic enzymes. The activity of the above enzymes decreases in some organs at the death caused by 2-15-minute hypoxia. Long-term intermittent adaptation to hypobaric hypoxia lowers the activity of glutathione peroxidase, -transferase and -reductase. The biological value of the two types of enzymatic responses may be different: stress-induced activation of glutathione metabolic enzymes can enhance resistance to stress and xenobiotics; however, their inhibition during hypoxic adaptation may produce the opposite effect.     

Influence of chemical treatments on glutathione S-transferases of maize with activity towards metolachlor and cinnamic acid

The subcellular distribution of glutathione S-transferase (GST) activity extracted from shoots of 3-day-old etiolated seedlings of maize (Zea mays L., Northrup-King 9283 hybrid) and the induction of soluble and membrane-bound GST activity by the safener benoxacor, the herbicide metolachlor and their combination (CGA-180937) were investigated. GST activity extracted from maize shoots was detected in both cytosolic and microsomal fractions and utilized 1-chloro-2,4-dinitrobenzene (CDNB), metolachlor, and trans-cinnamic acid (CA) as substrates. Soluble GST activity extracted from maize shoots was greater than microsomal with CDNB or metolachlor as substrate. Membrane-bound GST activity was greater than soluble with cinnamic acid as substrate. Washing the microsomal preparations from maize shoots with Triton X-100 increased GST(CA) activity. Pretreatment with the safener benoxacor or a formulated combination of the herbicide metolachlor with benoxacor induced soluble GST(CDNB), GST(metolachlor) and GST(CA) activities in maize shoots. Benoxacor and CGA-180937 induced also membrane-bound GST(CDNB) and GST(CA) activities in maize shoots, but did not affect membrane-bound GST(metolachlor) activity. These results confirm that maize contains multiple GST isozymes that differ in their substrate specificity and inducibility by safeners or other chemicals.     

Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.     

Immunolocalisation of the glutathione S-transferases, GST-26 and GST-28, within adult Schistosoma japonicum

This paper describes the first ultrastructural immunolocalisation study of the 26-kDa and 28-kDa glutathione S-transferases within adult Schistosoma japonicum (GST-26 and GST-28). Polyclonal antibodies raised against GST-28 (in mice) and against GST-26 (in rabbits) were used to examine the distribution of the proteins within adult parasites. Both proteins were localised within the parenchymal region of the male parasite. Additionally, both proteins were present within parenchymal cells located between the vitelline glands of female parasites. There were no detectable levels of GST-26 or GST-28 on the surface or within the tegument matrix of either the male or female worms. Possible functions for GST-26 and GST-28 within S. japonicum and their significance as vaccine target molecules are addressed.     

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.     

The effect of short term administration of cordafen on lipid peroxidation, activity of certain antioxidant enzymes and levels of reduced glutathione in erythrocytes

After 3 days of orally administrated cordafen (nifedipine) (30 mg per day divided in 3 equal doses) we revealed in red blood cells: statistically significant decrease of malonyl dialdehyde concentration, statistically significant increase of selenium-dependent glutathione peroxidase activity and superoxide dismutase activity. Also increased mean reduced glutathione concentration in erythrocytes of human peripheral blood was found, though statistically nonsignificant.     

Simultaneous protective effect of a new chelating agent and zinc, on the carbon tetrachloride induced hepatic injury in squirrels

X-ray test of the osteo-articular system of upper limbs was carried out on 1231 saweyers and 272 controls as well as 50 intellectuals. Three x-ray tests were carried out on 207 sawyers and on a control group of 95 workers, to assess the progression of osseous changes. The same types od changes were found in sawyers and control group workers; besides similar frequency in the examined parts of the upper limb was found in them. The most frequent changes are: osseous cyst, osteoporosis degenerative-deformative changes and calcar of the ulnar. The greatest amounts of changes were observed in the bones and joints of the hand and next in elbow joint and acromioclavicular joint. It was observed that amoungst the sawyers and the control group workers, the greatest amounts of changes occur after 2 years of work. Progression of osseous changes after 3-4 years of work was observed in 30,9% sawyers and 12,6% control group workers.     

Reversal of the sex difference in serum leptin levels upon cross-sex hormone administration in transsexuals

Women have higher circulating leptin levels than men. This sex difference is not simply explained by differences in the amount of body fat and is possibly influenced by their different sex steroid milieus. This prompted us to study prospectively the effects of cross-sex steroid hormones on serum leptin levels in 17 male to female transsexuals and 15 female to male transsexuals. Male to female transsexuals were treated with 100 micrograms ethinyl estradiol and 100 mg cyproterone acetate (antiandrogen) daily, and female to male transsexuals received testosterone esters (250 mg/2 weeks, im). Before and after 4 and 12 months of cross-sex hormone treatment, serum leptin levels and measures of body fatness were assessed. Before treatment, female subjects had higher serum leptin levels than male subjects independently of the amount of body fat (P < 0.01). Cross-sex hormone administration induced a reversal of the sex difference in serum leptin levels. Estrogen treatment in combination with antiandrogens in male subjects increased median serum leptin levels from 1.9 ng/mL before treatment to 4.8 ng/mL after 4 months and 5.5 ng/mL after 12 months of treatment (P < 0.0001). Testosterone administration in female subjects decreased median serum leptin levels from 5.6 to 2.6 ng/mL after 4 months and to 2.5 ng/mL after 12 months (P < 0.0001). Analysis of covariance revealed that the changes in serum leptin levels were independent of changes in body fatness in both groups (P < 0.01). In conclusion, these results indicate that sex steroid hormones, in particular testosterone, play an important role in the regulation of serum leptin levels. The prevailing sex steroid milieu, not genetic sex, is a significant determinant of the sex difference in serum leptin levels.     

Effect of tocopherol deficiency and additional administration of tocopherol, dibunol and disodium selenite on enzyme activity in rat liver, which take part in the biotransformation of nitrosodimethylamine

The alimentary tocopherol deficiency is accompanied by decreased hydroxylase, demethylase, NADH- and NADPH-reductase, aldehyde dehydrogenase, arylesterase and glutathione reductase activity in rat's liver. It decreased the reduced glutathione and increased it's oxidized form concentration in the tocopherol deficient animals. The stability of microsomal membrane is decreased to solubilizing action of deoxycholate and trypsin. This changes, possibly, caused elevation of alteration of function enzyme's and microsomal membrane after nitrosodimethylamine (NDMA) administration in deficient rats. The 7-days injection of tocopherol (20 and 100 mg/kg), dibunol (80 mg/kg), sodium selenite (30 mkg/kg) increased aldehyde dehydrogenase, esterase, glutathione-dependent enzymes activity and increased of reduced glutathione concentration in liver, suppressed lipid peroxidation and increased survival rats after lethal dose carcinogen treatment. Supplementation of tocopherol decreased harmful action of nitrosodimethylamine on microsomal membrane and enzymes activity.     

Hepatoprotective effect of C-phycocyanin: protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicty in rats

Effect of C-phycocyanin (from Spirulina platensis) pretreatment on carbontetrachloride and R-(+)-pulegone-induced hepatotoxicity in rats was studied. Intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of phycocyanin to rats, one or three hours prior to R-(+)-pulegone (250 mg/kg) or carbontetrachloride (0.6 ml/kg) challenge, significantly reduced the hepatotoxicity caused by these chemicals. For instance, serum glutamate pyruvate transaminase (SGPT) activity was almost equal to control values. The losses of microsomal cytochrome P450, glucose-6-phosphatase and aminopyrine-N-demethylase were significantly reduced, suggesting that phycocyanin provides protection to liver enzymes. It was noticed that the level of menthofuran, the proximate toxin of R-(+)-pulegone was nearly 70% more in the urine samples collected from rats treated with R-(+)-pulegone alone than rats treated with the combination of phycocyanin and R-(+)-pulegone. The possible mechanism involved in the hepatoprotection is discussed.     

Effect of the separate and combined administration of mestranol and phenobarbital on the development of altered hepatic foci expressing placental form of glutathione S-transferase in the rat

The stages in the carcinogenesis process include initiation, promotion and progression. Although many characteristics of tumor promotion and promoting agents have been reported, relatively few studies on the effects of combinations of promoting agents have been detailed. For study of the combined effects of phenobarbital (PB) and mestranol (MS) in multistage rat hepatocarcinogenesis, an initiation-promotion protocol was developed. Female rats were injected i.p. at 5 days of age with either diethylnitrosamine (DEN) (10 mg/kg) or the solvent tricaprilyn. At weaning, approximately 10 rats from both the DEN-initiated and the solvent control groups were provided basal diet alone, PB (10, 100 or 500 mg/kg diet), MS (0.02 or 0.2 mg/kg diet) or various combinations of both PB and MS in the basal diet. At 8 months of age, the rats were killed and the livers removed, sectioned and fixed in ice-cold acetone. Sections of 5 microgram thickness were stained for placental glutathione S-transferase (PGST) expression, and the volume fraction of liver occupied by altered hepatic foci was determined by stereology. In addition, incorporation of bromodeoxyuridine into nuclei of PGST-expressing (focal) and non-PGST-expressing (non-focal) hepatocytes was determined. Administration of the highest dose of PB resulted in a significant decrease in non-focal hepatocyte labeling index, with a 4-fold differential between the focal and non-focal hepatocyte labeling index. Administration of 0.2 mg/kg diet MS resulted in effective promotion. The non-focal labeling index was increased and the focal labeling index was further enhanced (3-fold) relative to the non-focal index by this dose of MS. Combination of the lower MS dose with PB resulted in at least an additive promoting effect; however, a lower volume fraction was noted for the combination of low MS dose plus the highest PB dose. Combination of the higher MS dose with PB resulted in an elevation of volume fraction only for the middle PB dose. These findings indicate that the potency of promotion by mixtures is modulated by the dose of each component as well as by pharmacodynamic and pharmacokinetic properties of each component of the mixture.