Haemolysis and artifactual lung damage induced by an euthanasia agent

The artifactual development of endothelial necrosis, pulmonary congestion, and oedema that has been reported in dogs, cats, and monkeys when the animals were killed with 1-1.5 ml/kg body weight of the euthanasia agent T-61 was reproduced in adult ewes of the merino breed. This species also exhibited a marked pulmonary congestion with intra-alveolar haemorrhages, septal oedema, and a diffuse cellular damage of the alveolar septa when the recommended dose of 0.3 ml/kg body weight was administered after forcing blood flow through one lung by clamping the contralateral hilum. The red coloration of the damaged lung areas may be due to haemolysis, another aspect of T-61 induced cell damage in this species. The degree of haemolysis increases with increasing blood concentration of the agent and approximates complete haemolysis at a T-61 blood concentration of 5%. The blood concentration dependent degree of haemolysis in sheep suggests a similar relationship between blood concentration of the agent and degree of pulmonary tissue damage.     

Potentiation of the hepatotoxic responses to chemicals in alloxan-diabetic rats.

Alloxan diabetes enhances the hepatotoxic response of male rats to chloroform and 1, 1, 2-trichloroethane, but not to trichloroethylene nor 1, 1, 1-trichloroethane. Insulin treatment partially protects the animals against the alloxan-induced enhancement of chloroform hepatotoxicity. Alloxan diabetes also enhances the hepatotoxic response to galactosamine but not to beryllium nor alpha-naphthylisothiocyanate.     

Potentiation of DNA damage and cytotoxicity by calmodulin antagonists

Many physical and chemical agents used in anti-neoplastic therapy are known to act by effecting DNA damage. The possibility that calmodulin may be an important modulator of the response to DNA damage in eukaryotic cells has been explored in a number of studies which use calmodulin antagonists in combination with agents known to damage DNA. We review these studies and discuss the therapeutic potential of calmodulin antagonists in combination therapy.     

Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer

DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks).     

Prolyl hydroxylase activity as an index of liver damage induced by ethanol

It was found that chronic intoxication of rats with ethanol results in an increase of prolyl hydroxylase activity in liver and serum of the experimental animals. The increase of enzyme activity precedes the morphological symptoms of liver damage. The possibility arises that the assay of prolyl hydroxylase in serum or in liver biopsy samples could be useful for the diagnosis of the tendency of some individuals to develop liver cirrhosis induced by ethanol.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

Potentiation by caffeine of ultraviolet-light damage in cultured human cells.

Five cultured human cell lines (T-1 kidney, Chang liver, H.Ep. No. 2, HeLa-S3 and HeLa-O) were irradiated with ultraviolet light and immediately exposed to 1.0 and 3.0 mM caffeine for 44 h thereafter. This caffeine treatment reduced the surviving fraction (assayed by colony formation) of the irradiated population, but did not significantly reduce the colony-forming ability of unirradiated control cells. These findings suggest that many cultured human cell lines exhibit post-UV potentiation of potentially lethal damage by caffeine.     

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 microg MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 microg g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 microg g(-1) dry weight at 48 h, indicating a depuration of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis.     

Protective effect of quercetin on liver damage induced by biliary obstruction in rats

The aim of this study was to evaluate the possible protective effects of quercetin (QE) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. A total of 24 male Wistar albino rats were divided into three groups: control, bile duct ligation (BDL) and BDL + received QE; each group contain 8 animals. The rats in QE treated groups were given QE (15 mg/kg) once a day intraperitoneally for 4 weeks starting 3 days prior to BDL operation. The changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with QE attenuated alterations in liver histology. The alpha smooth muscle actin (α-SMA), transforming growth factor beta (TGF-β1) positive cells and the activity of TUNEL in the BDL were observed to be reduced with the QE treatment. The data indicate that QE attenuates BDL-induced cholestatic liver injury, bile duct proliferation, and fibrosis. The hepatoprotective effect of QE is associated with antioxidative potential.     

Dimethylthiourea protects against mitochondrial oxidative damage induced by cisplatin in liver of rats

Cisplatin is one of the most effective chemotherapeutic agents. However, at higher doses liver injury may occur. The purpose of this study was to explore whether the hydroxyl radical scavenger dimethylthiourea (DMTU) protects against cisplatin-induced oxidative damage in vivo and to define the mitochondrial pathways involved in cytoprotection. Adult male Wistar rats (200–220 g) were divided into four groups of eight animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml/100 g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p), followed by 125 mg/kg body weight, i.p. (twice a day) until sacrifice. The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU + cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 h after the treatment). DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial damage in liver, therefore preventing elevation of AST and ALT serum levels. DMTU protected against (a) decreased hepatic ATP levels; (b) lipid peroxidation; (c) cardiolipin oxidation; (d) sulfhydryl protein oxidation; (e) mitochondrial membrane rigidification; (f) GSH oxidation; (g) NADPH oxidation; (h) apoptosis. Results suggest that antioxidants, particularly hydroxyl radical scavengers, protect liver mitochondria against cisplatin-induced oxidative damage. Several mitochondrial changes were delineated and proposed as interesting targets for cytoprotective strategy.     

Potentiation and suppression of mouse liver cytochrome P-450 isozymes during the acute-phase response induced by bacterial endotoxin

Infection and inflammation are known to affect the metabolism and disposition of drugs and carcinogens. We report a detailed study of the effects of bacterial endotoxin on the constitutive and inducible expression and activities of cytochrome P-450 isozymes from families P-450I, P-450IIB, P-450IIC and P-450III. In general high doses of high endotoxin caused very marked suppression of P-450 isozymes and associated activities. However, this effect was differential, the expression of certain isozymes being only slightly reduced whereas others were suppressed to almost undetectable levels. Low doses of endotoxin also gave differential effects on cytochrome P-450 expression. Of particular interest was the very marked potentiation of the inductive effect of both 3-methylcholanthrene and phenobarbital. In the case of 3-methylcholanthrene the 10-fold induction of activity was increased to 24-fold by concomitant endotoxin administration. In this regard it was interesting that 3-methylcholanthrene was an effective inducer of a wide variety of acute-phase proteins including metallothionein, serum amyloid A, fibrinogen and hemopexin. These data show that endotoxin, and therefore bacterial infection and inflammation, can have profound and differential effects on components of the cytochrome-P-450 monooxygenase system which could result in significant changes in susceptibility to the effects of drugs, chemical toxins and carcinogens.     

Low-dose gamma-ray irradiation reduces oxidative damage induced by CCl4 in mouse liver

We examined the effects of irradiation (50 cGy of gamma-ray) reducing the oxidative damage in carbon tetrachloride (CCl4)-hepatopathy mice. We made pathological examinations and analyzed transaminase activity (glutamic oxaloacetic transaminase and glutamic pyruvic transaminase), lipid peroxide level and the activities of endogenous antioxidants in the mouse. The irradiation was found to accelerate the recovery. Based on pathological examination as well as changes in each transaminase activity and lipid peroxide levels, it was shown that hepatopathy improved 3 d after the irradiation. The activities of glutathione reductase and glutathione peroxidase rapidly elevated after irradiation, and the total glutathione content gradually increased in the irradiation group. Both activities of gamma-glutamylcysteine synthetase and catalase were higher than normal at all times after the irradiation and gradually increased. In addition, the gamma-glutamylcysteine synthetase activity changed in a similar fashion to the total glutathione content. However, superoxide dismutase activity in both groups decreased and that of the irradiation group was significantly lower than that of the sham-irradiation group. These findings suggest that low-dose radiation relieved functional disorder at least in the liver of mice with active oxygen diseases.     

Caloric restriction prevents oxidative damage induced by the carcinogen clofibrate in mouse liver

Long-term caloric restriction in rodents is known to decrease levels of oxidative damage, which may contribute to an 'anti-ageing' effect. We show here that a shorter period (10 months) of caloric restriction had only small effects on levels of oxidative DNA and protein damage in the livers of mice, but completely attenuated increased oxidative damage caused by the carcinogen clofibrate. Since clofibrate is thought to exert its actions by increasing oxidative damage, our data suggest that 10 months of caloric restriction can increase the resistance of tissues to agents inducing oxidative stress. This may be an important factor in explaining how caloric restriction decreases cancer incidence.     

Quercetin protection against ciprofloxacin induced liver damage in rats

Ciprofloxacin is a common, broad spectrum antibacterial agent; however, evidence is accumulating that ciprofloxacin may cause liver damage. Quercetin is a free radical scavenger and antioxidant. We investigated histological changes in hepatic tissue of rats caused by ciprofloxacin and the effects of quercetin on these changes using histochemical and biochemical methods. We divided 28 adult female Wistar albino rats into four equal groups: control, quercetin treated, ciprofloxacin treated, and ciprofloxacin + quercetin treated. At the end of the experiment, liver samples were processed for light microscopic examination and biochemical measurements. Sections were prepared and stained with hematoxylin and eosin, and a histopathologic damage score was calculated. The sections from the control group appeared normal. Hemorrhage, inflammatory cell infiltration and intracellular vacuolization were observed in the ciprofloxacin group. The histopathological findings were reduced in the group treated with quercetin. Significant differences were found between the control and ciprofloxacin groups, and between the ciprofloxacin and ciprofloxacin + quercetin groups. Quercetin administration reduced liver injury caused by ciprofloxacin in rats. We suggest that quercetin may be useful for preventing ciprofloxacin induced liver damage.     

Chromosome damage induced by chemicals

During the past half century there has been a substantial increase in the number and extent of usage of drugs, food additives, pesticides and other environmental chemicals. As a result, mutation-induced susceptibility to disease, once largely self-eradicating, is now being conserved and even propagated. In addition, we have over the same time period, added to our internal and external environments, numerous synthetic chemicals, the cytogenetic properties of some of which have been reviewed, and to which man's metabolism has not necessarily been conditioned and adapted, and which may have the potential to augment the number of mutations of each succeeding generation.We need to know far more about what we are doing to ourselves and our planet, and the foetuses of our unborn children, and to the genetic heritage of these children, by the permissive use of the chemical wonders of our age. It would surely be impracticable that one should wait for time to reveal harmful mutations before we try to research out and eliminate the chemical mutagens.Chromosomal studies and evaluation of mutagenic potential of all new and established drugs, food additives and environmental chemicals should therefore be integral aspects of the current practices of safety evaluation of these materials, and the same stringent principles should be applied to the numerous chemicals met with as solvents and intermediates in industrial processes. Persons exposed to high risk, such as those working in chemical industries, or patients on continuous chemotherapy, should be offered routine chromosomal monitoring, and the appearance of adverse effects and abnormalities notified and correlated.     

Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis

Summary Steatohepatitis has recently been increasing as a cofactor influencing the progression of fibrosis, cirrhosis, adenoma and carcinoma in liver; however, the mechanisms by which it contributes to liver injury remain uncertain. We induced steatohepatitis in zebrafish embryos using thioacetamide (TAA). TUNEL assay revealed significant increasing of apoptosis in liver after 5 days post fertilization and the increasing of apoptosis was observed to be associated with the up-regulation of apoptotic genes such as, bad, bax, P-38a, caspase-3 and 8, and JNK-1. Histological sections by oil red O stain showed the accumulation of fatty droplets which causes the pushing of the nucleus towards one side. Up-regulation of steatosis markers such as, ACC, adiponectin, PTL, CEBP- and , SREBP-1 was also observed. Furthermore, the elevation of glutathione peroxidase in TAA treated embryos indicated that TAA induces lipid peroxidation which leads to causes liver damage. Zebrafish has already been considered as a good human disease model and in this context; TAA-treated zebrafish may serve as a good animal model to study the molecular pathogenesis of steatohepatitis. Moreover, non-availability of specific drugs to prevent steatohepatitis, this animal model may serve as a powerful preclinical platform to study the therapeutic strategies and for evaluating chemoprevention strategies for this disease.