2,3,7,8, tetrachlorodibenzo-p-dioxin induces oxygen activation associated with cell respiration.

We have investigated the influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on bioenergetic functions of isolated heart-mitochondria. Electron transfer and energy conservation activities were found to be decreased in the presence of very low amounts of the polychlorinated biphenyl compound (1.5 nmol/mg mitochondrial protein). The effect was greatest when substrates for complex I were used. In this case coupling of oxidative phosphorylation to respiration was drastically diminished, essentially at the expense of state 3 respiration, and P/O values were found around 2 instead of 3. Succinate-related energy conservation remained practically unaffected in the presence of TCDD, suggesting an interference of the toxic compound at coupling site I. SOD plus catalase were found to protect energy-linked respiration from the effect of dioxin indicating the involvement of superoxide radicals and H2O2 in the development of the observed phenomena. The present contribution provides experimental evidence on the formation of these oxygen species in the presence of TCDD. Furthermore, the site of action of TCDD is demonstrated and discussed in relation to the oxygen radical formation observed.

Physiologic function of the Wilson disease gene product, ATP7B.

The genes responsible for Wilson disease and Menkes syndrome have been cloned and identified as copper ATPases. These enzymes form part of a large family of transporters, the P-type ATPases. Although copper ATPases share strong structural similarities with these other pumps, comparatively little is known about their physiologic function. In this review, we examine data relating to the Wilson disease protein, ATP7B, in the liver. We present evidence suggesting that ATP7B is located intracellularly, together with data suggesting that, at least in part, ATP7B may also be found on the canalicular membrane. We also examine the form of copper that the transporter recognizes. We then review data on the Long-Evans Cinnamon rat, a model for Wilson disease, and discuss what effect the Wilson disease mutation has on copper transport. Finally, we conclude that, although we have made major advances in our understanding of copper metabolism in the liver, there are still many questions awaiting answers.

Responses of mitochondrial superoxide dismutase, catalase and glutathione peroxidase activities to aging.

The previous observation (Eur. J. Biochem., 82 (1978) 563--567) that age-dependent accumulation of lipid peroxides follows as a consequence of increased radical formation in mitochondria has prompted an examination of the response of a set of protective enzymes to the above situation. Levels of mitochondrial catalase activity as well as selenium-dependent glutathione peroxidase activity were found to be increased with age, while superoxide dismutase activity remained unchanged. No selenium-independent glutathione peroxidase activity could be detected either in preparations from young 3-month-old controls or in preparations from 2-year-old rats. Both the relatively high and unchanged levels of reduced glutathione and kinetic considerations suggest that glutathione peroxidase is preferentially involved in lipid peroxide metabolism, while catalase predominantly metabolizes mitochondrial H2O2.

Characterization of Cat-2t, a radiation-induced dominant cataract mutation in mice.

A dominant cataract mutation was detected recently among the offspring of x-ray-irradiated male mice. The mutation, which causes total lens opacity, has provisionally been designated by the gene symbol Cat-2t. In the lenses of heterozygous and homozygous Cat-2t mutants, the epithelial and fiber cells were swollen and the lens capsule was ruptured. The histologic analysis demonstrated a complete destruction of the cellular organization of the lens, which might be caused by its altered developmental processes. The data derived from biochemical investigations indicate that biochemistry of the cataractous Cat-2t lenses is affected: the osmotic state as indicated by the increased water content and increased Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity; the energy state as indicated by the decreased adenosine triphosphate (ATP) concentration; and the redox state as indicated by the enhanced content of oxidized glutathione. Additionally, the lenticular protein composition is altered because of the presence of vimentin in the water-soluble fraction. This cannot be explained by the enhanced crosslinking activity of transglutaminase. The changes of the osmotic, energy, and redox states are considered to be secondary in relation to the altered lenticular development. In contrast, the variations concerning vimentin and transglutaminase might be a biochemical indication of the changed development. Possible similarities to other dominantly expressed murine cataract mutants are discussed.

Assaying for hydroxyl radicals: hydroxylated terephthalate is a superior fluorescence marker than hydroxylated benzoate.

Generation of hydroxyl radicals in terephthalate (benzene-1,4-dicarboxylic acid) solution yields fluorescent 2-hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The efficiency, i.e. the relative increase of fluorescence per *OH radical, is about three times higher than that of the formation of salicylate (2-hydroxy-benzoate) from benzoic acid and approximately hundred-fold higher than that of the hydroxylation of phenylalanine. As the terephthalate molecule is symmetric with respect to ring-hydroxylation, only one isomer is formed; hence, mechanistic interpretation of the hydroxylation reaction is facilitated. The scavenging rate constant of terephthalate for *OH yielding the hydroxycyclohexadienyl adduct as first intermediate is close to the diffusion controlled limit (k = 3.3 x 10(9) M(-1) s(-1)). Therefore, competition of the detector molecule with biomolecules being present under physiological conditions is expected to be efficient. The assay can be used to detect 'free' *OH radicals produced by the radiolysis of water as well as 'hydroxyl analogous species' that have been suggested to arise from the interaction of complex-bound reduced metal with either oxygen or hydrogen peroxide, e.g. from Fenton reactions. Based on calibration with radiolytically generated hydroxyl radicals the detection limit of the method is estimated to be around 50 nmol/dm3. Terephthalate is classified non-toxic and hence may also prove useful for microdialysis and continuous flow experiments as observation of fluorescence is 'non-destructive' and the reporter substance does not necessarily have to be subjected to HPLC.

Stability of metallothionein in gastric juice.

Metallothionein (MT), is presumably the major Cd-binding component of human food. It is not or only partially destroyed by cooking. To study whether MT is stable in gastric juice MT was incubated at various pH values with pepsin and human gastric juice in vitro. Above pH 3.5 nearly all Cd remained bound to the protein and Cd-MT was resistant towards proteolysis. At pH values of 2.5 and 1.7 the protein was digested to 80% and 100%, respectively. At pH 2.5 two Cd-containing peptides with 25-30 amino acids similar to the alpha-domain of the protein were detected. At pH 1.7 these fragments were further degraded to smaller peptides. The results indicate that the pH-dependent proteolytic degradation of Cd-MT depends on the metal content of the protein. Furthermore, the identical results obtained with pepsin and human gastric juice suggest that Cd-MT proteolysis occurs in vivo and will affect Cd resorption.

UDP-glucuronosyl-, phenol sulfo-, and glutathione-S-transferase activities of mammalian cells in permanent culture.

Established cell cultures derived from mouse, rat, hamster, cat, and man were examined for the in vitro activities of UDP-glucuronosyl-, phenol sulfo-, and glutathione-S-transferases. The relative activities of the conjugation reactions differed considerably between the various cell lines: Glutathione-S-transferase activities were present in all cell lines with wide variation between different transferase forms. Phenol sulfotransferase was not detectable at all in most of the cell lines tested or was present at very low levels. In contrast, UDP-glucuronosyltransferase activity was expressed in the majority of cultures. Established cultures containing specific combinations of the various types of conjugases should be useful in examining their role in the inactivation and activation of potentially toxic chemicals.

D-penicillamine induces rat hepatic metallothionein.

Intraperitoneal injection of D-Penicillamine (D-PA) at a dose range of 20-500 mg/kg increased rat hepatic but not renal and pancreatic metallothionein (MT). Elevated MT predominantly contained Zn. Maximal induction was obtained 18 h after a single injection of 200 mg D-PA/kg resulting in 148 +/- 18 micrograms MT/g liver which was 16.4-times the control level of 9 +/- 2 micrograms MT/g. At 48 h after injection, MT declined to 18 +/- 9 micrograms MT/g liver. At maximal MT increase the content of total hepatic Zn but not of Cu was elevated. Increased amounts of Zn in liver homogenate, cytosol and MT could be detected approximately 4 h after injection of 200 mg D-PA/kg. Concomitantly there was a decrease in Zn bound to cytosolic non-MT ligands. All Zn changes reversed at 18 h. These data show that already single doses of D-PA cause induction of Zn-thionein in rat liver and lead to synchronous redistribution of Zn from endogenous sources to newly synthesized MT.

Hohenheim consensus workshop: copper.

Copper (Cu) is an essential trace element with many physiological functions. Homeostatic mechanisms exist to allow Cu to act as a cofactor in enzymatic processes and to prevent accumulation of Cu to toxic levels. The aim of this commentary is to better understand the role of dietary Cu supply in deficiency and under physiological and pathological conditions. The essentiality of Cu can be attributed to its role as a cofactor in a number of enzymes that are involved in the defence against oxidative stress. Cu, however, has a second face, that of a toxic compound as it is observed with accumulating evidence in hepatic, neurodegenerative and cardiovascular diseases. The destructive potential of Cu can be attributed to inherent physico-chemical properties. The main property is its ability to take part in Fenton-like reactions in which the highly reactive and extremely deleterious hydroxyl radical is formed. Diseases caused by dietary Cu overload could be based on a genetic predisposition. Thus, an assessment of risk-groups, such as infants with impaired mechanisms of Cu homeostasis regarding detoxification, is of special interest, as their Cu intake with resuspended formula milk may be very high. This implies the need for reliable diagnostic markers to determine the Cu status. These topics were introduced at the workshop by the participants followed by extensive group discussion. The consensus statements were agreed on by all members. One of the conclusions is that a re-assessment of published data is necessary and future research is required.

Glutathione and glutathione S-transferase activities of mammalian cells in culture.

Continuous cell cultures derived from various tissues of rat, mouse, hamster and man were assayed for their glutathione (GSH) content and glutathione S-transferase activities. GSH S-transferase activities were monitored toward the substrates 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (PO). All cell lines tested contained appreciable amounts of GSH ranging from 10 to 65 nmol/mg cellular protein. Likewise, all cell lines expressed GSH S-transferase activities. However, the various cell lines differed considerably in their relative transferase activities exhibiting some degree of species-specificity.

Metallothionein, cadmium, copper and zinc levels of human and rat tissues.

Metallothionein (MT), zinc (Zn), copper (Cu) and cadmium (Cd) were determined in 10 tissues (brain, heart, kidney cortex, liver, lung, muscle, pancreas, small intestine, spleen and stomach) from human autopsies (10 male individuals, mean age 43 +/- 9 years, all smokers) and Wistar rats. The mean tissue concentrations of MT in the human samples varied between 3.8 and 495 micrograms/g wet weight in spleen and kidney cortex, respectively. In most tissues human MT levels were high as compared to rats; particularly in liver and kidney cortex human MT levels exceeded those of rats about 25- and 10-fold, respectively. Positive linear relationships were observed between Zn or Cu and MT in human liver and between Cd and MT in human kidney cortex.

Metallothionein, copper and zinc in fetal and neonatal human liver: changes during development.

Total and cytosolic zinc (Zn) and copper (Cu), cytosolic metallothionein (MT) and the Cu-load of MT were investigated in fetal (22, 24 and 32 gestational weeks) and neonatal (2-15 months) human liver. Whereas the fraction of cytosolic Zn remained constant at 66% of the total independent of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to about 100% within 12 months postnatally. The MT content was higher in fetal than in neonatal liver. There was a linear correlation (r = 0.996) between cytosolic MT and Zn in both fetal and neonatal liver but not between MT and Cu. In contrast to fetal liver, the Cu-load of MT in neonatal liver seems to be determined by the Zn/Cu ratio in the cytosol. The results suggest that MT is involved in the regulation of Cu and Zn metabolism during fetal and neonatal development.

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione.

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538, and TA98 without metabolic activation. Presumably owing to conjugation with bacterial GHS, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae.

Glutathione-deficient (gsh-) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh-:GSH+ phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh- mutants belong to one complementation group.

Glutathione and glutathione S-transferases in the Salmonella mammalian-microsome mutagenicity test.

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH. The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.

Different pathomorphologic patterns in exogenic infantile copper intoxication of the liver.

Pathomorphology of the liver has been reviewed in 12 German infants with chronic exogenic copper intoxication. In 8 cases severe liver damage with diffuse accumulation of Mallory bodies and liver cell necrosis mimicking florid Indian childhood cirrhosis (ICC) was found. Seven of these children died because of liver failure. One child received liver transplantation at the age of 9 months. In contrast, 4 children with a stable clinical course had a complete micronodular cirrhosis in liver biopsy. The characteristic morphological features of ICC, especially ballooning of liver cells and accumulation of Mallory bodies, were only slightly expressed or even lacking. There was no correlation between the copper content of the liver and the severity of liver damage. The copper concentration varied between 541 micrograms/g dry weight (norm < 50 micrograms/g) and 2.154 micrograms/g dry weight in fatal cases. In surviving infants even higher concentrations of up to 698 micrograms/g fresh weight (norm < 5 micrograms/g), were found. The amount of free cytosolic copper varied between 900-4,900 ng/mg protein (13-70 times of normal). In conclusion, a spectrum of pathomorphological alterations exists in exogenic infantile copper disease which correlates with the clinical outcome in contrast to the copper content of the liver. Copper intoxication of the liver should be of diagnostic concern in any case of unclear micronodular cirrhosis in early infancy.

Threshold levels in toxicology: significance of inactivation mechanisms.

Metabolic inactivation of chemicals may prevent toxic effects of reactive intermediates when present at low levels whereas inactivation may be overcome at high levels changing dose-effect relation. This is demonstrated in various in vitro test systems: a) Monooxygenase-mediated metabolism causes formation of reactive oxygen species which induce DNA repair in lymphoblastoid cells. DNA damage is suppressed in the presence of glutathione (GSH), catalase or superoxide dismutase. b) Chloroprene is mutagenic in Salmonella typhimurium but not carcinogenic, possibly due to inactivation by GSH-conjugations. c) Chlorodinitrobenzene is not mutagenic is Salmonella typhimurium in the presence of GSH. However it is increasingly mutagenic at concentrations exceeding those of the GSH. d) Suppression of glucuronidation and sulfation in isolated hepatocytes highly increases irreversible binding of naphthalene. It is concluded that information on the metabolism of chemicals is essential for interpretation of toxicity studies in animals and their relevance to man.

Metallothionein induction by nonsteroidal antiinflammatory drugs.

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).