GST isoenzymes in matched normal and neoplastic breast tissue.

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore we investigated GST activity and the protein expression of glutathione S-transferases (GSTs) isoenzymes known to be involved in the metabolism of endogenous and exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. The interindividual variation in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and of 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) with glutathione (GSH) by cytosolic glutathione S-transferases (GSTs) were investigated in human breast matched normal and tumor samples. The GSTA, GSTM, GSTP and GSTT isoenzymes from the crude extracts of matched breast normal and tumor tissues in terms of their immunological properties using western blotting were compared. In most of the samples, the GST activities were higher in the tumor than in the normal cytosolic fractions against both CDNB and EPNP. In the western blotting analysis, it was proved statistically that in normal and tumor epithelial cells, there was difference between GST pi and theta isoenzymes expressions (p0.05). In normal epithelium there was a stronger GST theta expression than in invasive tumor tissues (p=0.013). However, the stronger GST pi expression was observed in tumor epithelium than in normal epithelium in human breast cancers (p=0.000). We found the GSTP protein level and GST activities were higher in the breast tumor than in the normal cytosolic fractions against both CDNB and EPNP, thus implicating a certain biological importance.

CYP and GST polymorphisms and survival in advanced non-small cell lung cancer patients.

UNLABELLED: Several polymorphisms in cytochrome P-450s (CYP)s and Glutathione S-transferases (GST)s have been reported to be associated with survival rates of patients with non-small cell lung cancer (NSCLC) but the studies in this regard are scarce and the results are contradictory. In this study, CYP1A1 (Ile462Val), CYP1B1(Asn453Ser), GST M1, GSTP1 exon 5 (Ile105Val) and exon 6(Ala114Val) and GSTT1 polymorphisms were determined in 138 patients with advanced NSCLC to evaluate their role in survival. Of the studied CYP and GST polymorphisms only GSTP1 exon 6 variant significantly altered (improved) the survival compared to wild type (p=0.036) with median survival of 22.2 months and 16.1 months, respectively. Multivariate analysis also revealed a significant reduction of adjusted hazard ratio of death associated only with the GSTP1 exon 6 variant genotype of 0.45 (95% confidence interval (95% CI), 0.23-0.89, p=0.022). These results show that the GSTP1 exon 6 variant genotype is associated with improved survival in the patients with advanced NSCLC. KEYWORDS: Cytochrome P-450, glutathione S-transferase, non small cell lung cancer, polymorphism, survival.

Expression of CYP and GST in human normal and colon tumor tissues.

We investigated the immunohistochemical staining characteristics of cytochrome P450 1A1 (CYP1A1), CYPB1, CYP2E1, and glutathione S-transferase P1 (GSTP1), GSTT1, GSTO1, GSTK1 in colon tumor and surrounding normal colon tissues. Tissues were obtained from 47 patients with colon adenocarcinoma and the staining intensity of tumor and control tissues was compared. CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTT1, GSTO1 and GSTK1 expressions in colon cancer cells were significantly greater than those in normal colon epithelial cells. No significant relation was found between the isoenzyme expressions and age, gender, smoking status, tumor grade and tumor stage. The higher expressions of CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTO1, GSTT1 and GSTK1 in tumor than in normal colon tissues may be important for colon cancer progression and development.

Comparison of NAT1, NAT2 and GSTT2-2 activities in normal and neoplastic human breast tissues.

In this study, arylamine N-acetyltransferases, NATs (E.C.2.3.1.5) and glutathione-S-transferase-T2-2, GSTT2-2 (E.C.2.5.1.18) enzyme activities in the breast tumor and surrounding tumor-free tissues of 22 female breast cancer patients with infiltrating ductal carcinoma were measured. The possible impacts of grade of malignancy, chemotherapy treatment, estrogen receptor status and menopausal status on all enzyme activities were evaluated. The results showed that, both NAT2 and GSTT2-2 display significant differences between tumor and tumor-free breast tissues, while no difference was observed in NAT1. Grade of malignancy seems to be positively associated with NAT1 and negatively associated with GSTT2-2. Though, both NAT2 and GSTT2-2 have increased mean tumor activities, the grade of malignancy, chemotherapy status, menopausal status or estrogen receptor status are not correlated statistically.

A study on the antioxidant activities of some new benzazole derivatives.

The in vitro antioxidant properties of some new benzazole derivatives (1-10) such as benzoxazoles, benzimidazoles, and benzothiazoles were determined by their effects on the rat liver microsomal NADPH-dependent lipid peroxidation (LP) level, the scavenging of superoxide anion and the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds 1, 2, 4 and 6, showed potent scavenging effect on superoxide radical at 10(-3) M. Compound 8, 5-nitro-2-(phenoxymethyl)benzimidazole, strongly inhibited lipid peroxidation at 10(-3) M concentration.

Acute effect of nebulized budesonide in asthmatic children.

The acute anti-inflammatory effects of inhaled steroids at high doses and their use at home and as emergency treatment of acute asthma attacks in children have been evaluated in many clinical studies. However very little is known about their additional bronchodilator response to systemic steroids plus nebulized salbutamol in the early management in children. Asthmatic patients aged between 5-15 years were investigated in a double-blind, placebo-controlled fashion. Both the study group (Group I) and the control group (Group II) received three consecutive doses of nebulized salbutamol (0.15 mg/kg/dose) and one dose of parenteral methylprednisolone (1 mg/kg/dose, intramuscularly). After this treatment, nebulized budesonide (1 mg/dose) was administered to patients in the study group and placebo (nebulized saline) was administered to patients in the control group. Pulmonary index scoring and peak flow meter was performed to both groups before and after the treatment. There were twelve patients in Group I (mean age: 7.90 +/- 2.34 years) and fourteen patients in Group II (mean age: 9.36 +/- 2.55 years). There was no difference between the two groups with respect to age (p = 0.1421), gender (p = 1.000) and inhaled steroid prophylaxis rate (p = 0.2177). No statistically significant difference was detected between the two groups with respect to the pulmonary index score (p = 0.3528). Yet, there was a statistically significant difference between the two groups with respect to the increase in PEFR (p = 0.0155). The positive acute effect of nebulized budesonide in addition to systemic steroids and nebulized salbutamol in improving the spirometric indices in asthmatic children is an encouraging finding for further investigations of its routine use in the pediatric emergency department.

Genotoxicity studies on benzimidazole retinoids.

Retinoids consist of a family of naturally occuring compounds including all-trans retinoic acid (ATRA), retinal, retinol (vitamin A), 9-cis retinoic acid, 13-cis retinoic acid as well as a large number of synthetic derivatives. Retinoids are known to elicit diverse pharmacological profiles such as controlling cell differentiation/proliferation and modulating specific premalignant lesions and reducing second primary tumors in patients. Clinical use of retinoids is limited due to their toxicity. Three benzimidazole retinoid derivatives (BITN, BITNm, BITNe) were synthesized and were examined in terms of genotoxicity towards human lymphocyte cultures by sister chromatid exchange (SCE) analysis. It has been found that BITN decreased the number of SCEs 20% at 10(-6) M, but had no effect at 10(-5) M. No significant effect on SCEs was observed for BITNm and BITNe at both concentrations. ATRA increased the SCEs (35%) at 10(-5) M but had no effect at 10(-6) M. The results have shown that benzimidazole retinoids did not induce SCE significantly. Besides this, BITN reduced the SCEs and had a protective effect at low concentration. Since the induction of glutathione S-transferase (GST) is associated with anticancer drug resistance, the effects of BITN, BITNm, BITNe and ATRA on human lymphocyte GSTs were also investigated using CDNB as substrate. BITN and BITNm induced GST activities 54% and 49% respectively at 10(-5) M, but had no effect at 10(-6) M. BITNe induced GST activity 62% at 10(-5) M and 35% at 10(-6) M. ATRA had no effect on GST activity at 10(-5) M.

Molecular characterization of zeta class glutathione S-transferases from Pinus brutia Ten.

Glutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants.In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1.

cis-platinum-mediated decrease in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis.

Previously we had shown that cis-platinum decreases testosterone levels in rat serum and that hCG reverses this effect. The purpose of these studies was to determine the biochemical basis of cis-platinum-mediated effects on testicular testosterone production. In the testis of rats treated with cis-platinum (7 mg/kg, iv), the mitochondrial P-450scc concentration and side-chain cleavage activity were depressed by 40%. Also, the microsomal 17 alpha-hydroxylase activity and cytochrome P-450 concentration were decreased. Testicular binding capacity (in vitro) for [125I]hCG was decreased by 75-80%. On the other hand, FSH binding to Sertoli cell membrane receptors was not appreciably changed. hCG (25 IU/100 g daily) in treated rats caused complete occupancy of the remaining 20-25% LH receptors and caused a 20- to 30-fold increase in serum and testicular testosterone, a 2-fold increase in mitochondrial P-450scc, and a 5-fold acceleration of side-chain cleavage activity. 17 alpha-Hydroxylase activity and microsomal cytochrome P-450 were not increased over the control values. In addition to testicular functions, pituitary glycoprotein hormone production was assessed. Treatment of rats with cis-platinum (7 mg/kg, iv) did not change serum LH or FSH, but caused a 50% decrease in serum and testicular testosterone levels. A GnRH challenge test (1.5 micrograms/100 g, in 30 min) of treated rats caused prompt increases of 10- to 15-fold in serum LH and resulted in increases in serum and testicular testosterone. Thus, there was little evidence for cis-platinum effects at the level of hypothalamus or pituitary that could account for the decreased testosterone production. Reversal of the cis-platinum effect on steroidogenesis by hCG or GnRH appears to be due to the induction of suprasaturating levels of LH with full occupancy of remaining Leydig cell LH receptors. This, in turn, would reverse the diminished levels of mitochondrial side-chain cleavage activity and cytochrome P-450scc. These data suggest that cis-platinum causes a depression in serum testosterone, mainly by decreasing the number of LH receptors and inhibiting side-chain cleavage activity.

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.

Inhibition of human hepatic and placental xenobiotic monooxygenases by imidazole antimycotics.

Three imidazole antimycotic drugs, ketoconazole, clotrimazole and miconazole, were studied to characterize the inhibition of aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (ECDE) and 7-ethoxyresorufin O-deethylase (ERDE) activities in human liver and placenta in vitro in comparison with liver enzymes from control, phenobarbital (PB) and 3-methylcholanthrene (MC) pretreated rats. All three compounds inhibited rat liver enzymes, although MC pretreatment seemed to lead to a resistance of inhibition relative to PB-treated and control animals. There were large differences in the extent of inhibition of human hepatic and placental activities. Furthermore, while the type of inhibition of the hepatic ERDE was competitive or mixed, that of the placental enzyme cannot be described in ordinary terms of inhibition kinetics. Ketoconazole and clotrimazole were relatively potent inhibitors of maternal cigarette smoking-induced placental ECDE activities (IC50 values from 0.5 microM to 5 microM), whereas much less inhibition of the placental AHH activity was obtained with ketoconazole and miconazole (IC50 values from 50 microM to 500 microM). In most cases, hepatic enzymes were less sensitive to antimycotics than placental activities. This was in contrast with results from rat enzyme studies, in which MC pretreatment seemed to decrease the inhibitory response.

Differential combined effect of cadmium and nickel on hepatic and renal glutathione S-transferases of the guinea pig.

When male guinea pigs were given a single dose of Cd (2.0 mg Cd2+/kg, ip) 72 hr prior to sacrifice, the hepatic reduced glutathione (GSH) level did not change although glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), and 1,2-epoxy-3-(p-nitrophenoxy) propane (ENPP) increased significantly as compared to controls. Cd did not change the renal GSH level and GST activities toward CDNB and EAA. However, significant increase was observed in the GST activity for DCNB whereas GST activity for ENPP was significantly inhibited by Cd. When the animals were given a single dose of Ni (14.8 mg Ni2+/kg, sc) 16 hr prior to sacrifice, significant increases were observed in hepatic GSH level and GST activities toward CDNB, DCNB, EAA and ENPP. Ni, however, depressed the renal GSH level and GST activities toward CDNB, DCNB and ENPP significantly. The renal GST activity toward EAA remained unaltered. For the combined treatment, guinea pigs received the single dose of Ni 56 hr after the single dose of Cd and then they were killed 16 hr later. In these animals, no significant alteration was observed in the hepatic GSH level. The augmentation of elevation was observed in hepatic GST activities toward CDNB and DCNB. Combined metal treatment did not potentiate the elevation of hepatic GST activities toward EAA and ENPP to any greater degree. The depression of renal GSH level was significantly ameliorated by the combined treatment. Combination treatment potentiated the depression of renal GST activity for ENPP but not for CDNB.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effects of ethanol and cigarette smoke on hepatic and pulmonary xenobiotic metabolizing enzymes in rats.

The combined effects of ethanol (EtOH) and cigarette smoke (CS) on hepatic and pulmonary monooxygenase (MO) activities (aniline 4-hydroxylase (AH), aminopyrine N-demethylase (AMND), 7-ethoxyresorufin O-deethylase (EROD), p-nitroanisole O-demethylase (p-NAOD)), lipid peroxidation (LP) and reduced glutathione (GSH) levels and glutathione S-transferase (GST) activities toward several substrates (l-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP)) were determined and compared with those of EtOH or CS alone in rats. When the male adult rats (225-275 g) were treated with 10% EtOH (v/v) in their drinking for 21 days AH, AMND and EROD activities and LP and GSH levels increased significantly whereas GST activity for EAA decreased significantly in liver as compared to controls. EtOH did not change the hepatic p-NAOD and GST activities toward CDNB, DCNB and ENPP. In lung, EtOH increased GST activities toward CDNB and ENPP and LP level but decreased GST activity toward DCNB, significantly. No alterations were noted in pulmonary MO activities and GST activity toward EAA and GSH level by EtOH treatment. When the animals were exposed to CS five times a day, with 1 h intervals, for 3 days in a chamber where smoke and fresh air lead alternatively, AMND, EROD and p-NAOD activities, GST activity toward EAA and GSH level increased but LP level and GST activity for ENPP decreased significantly in liver. CS did not alter the hepatic AH and GST activities toward CDNB and DCNB. In lung, CS increased AH, EROD and p-NAOD activities and LP and GSH levels and decreased all the GST activities studied significantly. CS had no influence on pulmonary AMND activity. For the combined treatment, the animals were treated with 10% EtOH (v/v) in their drinking water for 21 days and during the last 3 days they were exposed to CS five times a day, with 1 h intervals, in a chamber where smoke and fresh air lead alternatively. In these animals, augmentation of elevations were noted in AH and p-NAOD activities and LP and GSH levels but not in EROD and AMND activities in liver. Combined treatment significantly decreased GST activity toward CDNB, ameliorated the alteration caused by either EtOH or CS treatment alone on GST activity toward EAA and potentiated the depression of GST activity toward ENPP to a greater degree. No change was observed in GST activity toward DCNB. In lung, combined treatment potentiated the elevations of AMND and p-NAOD activities and LP level and not those of AH and EROD activities. GST activities toward CDNB, DCNB and ENPP were highly elevated by the combined treatment. No changes were observed in pulmonary GSH level and GST activity for EAA by the combined treatment. These results reveal that the regulations of the hepatic and pulmonary MO and GST are differentially influenced by EtOH, CS and the combined treatment.

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate.

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.

A study on the antioxidant capacities of some benzimidazoles in rat tissues.

Seven benzimidazole compounds were synthesized and their in vitro effects on rat liver, lung and kidney microsomal NADPH-dependent lipid peroxidation (LP) levels were determined. The significant decrease in male rat liver microsomal LP level was noted only by the compound 4 at 10(-4) M (20%) and 10(-3) M (40%) concentrations whereas the other compounds were ineffective. In lung, only the compound 6 at 10(-4) M concentration exhibited significant alteration, i.e. 56% increase, in LP level. In kidney, however, apart from the compound 4, all the compounds increased LP level (35-52%) significantly. The classical antioxidant, butylated hydroxy toluene (BHT), at 10(-4) M concentration, significantly decreased LP level about 70%, in all the tissues studied. To clarify the effects of compounds 4 and 6 on LP, the responses of some CYPs, which are active in producing reactive oxygen species, to these compounds were also investigated. The compound 4 at 10(-4) and 10(-3) M concentrations inhibited the hepatic microsomal ethoxyresorufin O-deethylase (EROD) (37 and 65%) and pentoxyresorufin O-depenthylase (PROD) (14 and 62%) enzyme activities significantly. However, it did not alter the hepatic microsomal NADPH-cytochrome P450-reductase activity. BHT, at 10(-3) M concentration, significantly inhibited hepatic microsomal EROD (73%), PROD (62%) and NADPH-cytochrome P450 reductase (17%) enzyme activities. Caffeine (10(-3)M) and SKF 525A (10(-3)M), which are specific inhibitors of EROD and PROD enzyme activities, significantly decreased the enzyme activities 33 and 77%, respectively. Caffeine was unable to alter hepatic microsomal NADPH-cytochrome P450 reductase enzyme activity whereas SKF 525A significantly inhibited (80%) it. In lung and kidney, the compound 6 at 10(-4)M concentration significantly increased EROD (44 and 19%) and PROD (103 and 86%) enzyme activities. However, the elevation of PROD enzyme activity in both tissues was observed to be more pronounced than that of EROD enzyme activity. This compound was ineffective on lung and kidney microsomal P450-reductase enzyme activity. These results reveal that the synthesized benzimidazoles have variable tissue dependent in vitro effects on LP due to their distinct effects on CYP activities but not on NADPH-cytochrome P450 reductase activity in rats.

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes.

When male rats were given either a single dose of cadmium (3.58 mg CdCl2.6H2O/kg, i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl2.6H2O/kg, s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg/kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg/kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.

DNA single-strand breakage in rat lung, liver and kidney after single and combined treatments of nickel and cadmium.

Single-strand breaks were observed in rat lung and kidney after acute treatment of animals with CdCl2 (4 mg/kg body weight) injected intraperitoneally and NiCl2, (44.4 mg/kg body weight) injected subcutaneously. In the rat liver, no single-strand breakage was evident with those doses in single and combined metal treatments. The most susceptible tissue in rats to cadmium or nickel chloride treatment was the lung tissue. The single-strand breaks were higher in cadmium treatment than in nickel treatment in the rat lung. Also the response to cadmium treatment was obtained earlier than nickel. Rat kidney was also responsive to cadmium treatment. However, the response, although statistically significant, was much lower than the one obtained in rat lung. The combined treatment, which was done by administrating cadmium prior to nickel administration, reduced the number of single-strand breaks significantly and reversed them to control values in rat lung and kidney. This study confirms that cadmium and nickel create single-strand breaks when administered alone in the rat lung. This effect, which was seen in the single metal treatments, was reduced in the combined treatments.

Sister chromatid exchanges in human lymphocytes treated in vitro with cadmium in G(o) and S phase of their cell cycles.

Sister chromatid exchanges (SCEs) were analyzed in human phytohemagglutinin-activated peripheral lymphocyte cultures exposed to varying concentrations (10(-7)-10(-3) M) of cadmium chloride in vitro at two different stages of the cell cycle, G(o) and early S phase. When cadmium chloride was administered at the G(o) phase, no increase in the SCEs were observed for the doses 10(-6) and 10(-5) M. Concentrations equal to or larger than 10(4) M cadmium chloride were lethal to human lymphocytes in our experimental conditions. A highly statistically significant increase was observed in the SCE frequency with increasing cadmium chloride concentration (10(-7)-10(-4)) when cadmium was administered at the early S phase, which was 24 h after culture initiation. The increase in SCE frequency was higher when the cultures were terminated at 54 h, compared to termination at 72 h. In order to examine the effects of cadmium administered at the S phase on SCE frequency in different individuals, 10(-5) M concentration was used and the cultures were terminated at 54 h after culture initiation. A 2- to 3-fold increase in the SCE frequency was observed in all six individuals examined. A progressive decrease in the proliferative index was also observed by increasing cadmium chloride concentration. These results demonstrate that the genotoxicity of cadmium chloride may be changed depending on the stage of the cell cycle in human lymphocytes. This may be one of the reasons of contradictory findings in the literature.

In vitro effects of cadmium and nickel on glutathione, lipid peroxidation and glutathione S-transferase in human kidney.

The in vitro effects of cadmium chloride (CdCl(2)) and nickel chloride (NiCl(2)) on reduced glutathione (GSH) level, lipid peroxidation (LP) and glutathione S-transferase (GST) activity towards the substrate l-chloro-2,4-dinitrobenzene (CDNB) in human kidney 10,000 g supernatant were examined. For comparative purposes similar studies were also performed using rat kidney 10,000 g supernatant. No alteration was observed in GSH level of human or rat kidney as a result of either CdCl(2) or NiCl(2) at the concentrations studied (10(-3)-10(-7)m). In human kidney 10,000 g supernatant, CdCl(2) and NiCl(2) were not able to alter the LP at the concentrations studied. Similarly, apart from a slight decrease at a NiCl(2) concentration of 10(-3)m, no effect of either CdCl(2) or NiCl(2) was noted on the LP of rat kidney 10,000 g supernatant. In human kidney 10,000 g supernatant, CdCl(2) at high concentrations (10(-4) and 10(-3)m) inhibited the metabolism of CDNB by GST, whereas in rat kidney 10,000 g supernatant the enzyme was inhibited in a concentration-dependent manner by CdCl(2). The degree of inhibition was similar in kidney 10,000 g supernatants of both human and rat, with 10(-4)m (about 28% inhibition) and 10(-3)m (about 44% inhibition) CdCl(2). NiCl(2), however, did not affect the metabolism of CDNB by GST in either human or rat kidney 10,000 g supernatant.

Production of superoxide radicals by sheep liver nitrofurantoin reductase: In vitro effects of nitrofurantoin metabolites on DNA.

The reduction of nitrofurantoin by purified liver nitrofurantoin reductase was followed by the production of superoxide radicals, which were detected by the reduction of epinephrine. The conditions for the formation of superoxide radicals were optimized. The maximum superoxide radical formation occurred at approximately 3.5 mug purified reductase, with optimum pH of 7.8 and at 0.05 mm nitrofuran- toin concentration. The K(m) and V(max) values were calculated as 1.95 x 10(-2) mm and 4.81 nmol Superoxide formed per minute. An obliteration of the banding pattern was observed on the agarose gel electrophoresis of sheep liver DNA, which was incubated in the nitrofurantoin-nitrofurantoin reductase system, as a possible consequence of the generation of superoxide radicals during the reduction of nitrofurantoin by nitrofurantoin reductase.