NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I.

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)-the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1⁻/⁻, Cyp1a2⁻/⁻ and Cyp1a1/1a2⁻/⁻ knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Role of P450 1A1 and P450 1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: studies in Cyp1a1-/-, Cyp1a2-/-, and Cyp1a1/1a2-/- mice.

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Angiotensin-converting enzyme gene polymorphism and N-Acetyl-ß-D-glucosaminidase excretion in endemic nephropathy.

BACKGROUND: Tubular proteinuria and enzymuria are hallmarks of endemic nephropathy (EN). The role of I/D angiotensin convertase (ACE) gene polymorphism has not yet been elucidated in this peculiar chronic tubulointerstitial nephritis, and our aim was to investigate the role of this polymorphism in EN focusing on the urinary N-acetyl-ß-D-glucosaminidase (NAG) excretion, a biomarker of proximal tubular damage. METHODS: ACE genotype and allele frequencies were determined in 229 farmers (147 women and 82 men) from an endemic Croatian village. The farmers were stratified according to the WHO criteria into the following subgroups: those 'at risk' for EN (n = 37), 'suspected of having EN' (n = 57), and 'others' (n = 135). RESULTS: There were 74 (32.3%) subjects homozygous for the D allele, 99 (43.2%) heterozygous (ID genotype) and 56 (24.4%) homozygous for the I allele. No differences in allele frequency were found between the established WHO subgroups (p > 0.05). In the whole group, DD subjects had significantly higher values of diastolic blood pressure (p = 0.003) and urinary NAG than subjects with ID and II genotype (5.5 ± 1.2 vs. 4.0 ± 3.0 vs. 3.8 ± 4.2, respectively; p = 0.023). The highest values of serum creatinine (p = 0.02), proteinuria (p = 0.03) and urinary NAG (6.0 ± 3.7 vs. 3.7 ± 2.1 vs. 3.0 ± 1.6, respectively; p = 0.008) were observed in those suspected of having EN group with the DD genotype. CONCLUSION: ACE gene polymorphism is not a risk factor for EN. However, it might influence the clinical course of EN, and increased excretion of NAG might be a prognostic marker of this chronic tubulointerstitial nephritis.

Local and Systemic Oxidative Stress in Balkan Endemic Nephropathy Is Not Associated with Xanthine Oxidase Activity.

Balkan endemic nephropathy (BEN) represents a chronic tubulointerstitial nephropathy which is followed by the progression of kidney fibrosis to end-stage kidney failure. The critical involvement of poisons in food (aristolochic acid (AA), ochratoxin, and heavy metals) and selenium deficiency are among nutritive factors which contribute to the pathogenesis of BEN, due to reactive oxygen species (ROS) liberation and/or decreased antioxidative defence system. The aim of the study is to distinguish a possible systemic and local origin of ROS through the measurement of xanthine oxidase (XO) activity in urine and plasma, along with the determination of the oxidative changes in lipids and proteins. The study included 50 patients with BEN and 38 control healthy subjects. We noted increased levels of both thiobarbituric acid-reactive substances (TBARS) and advanced oxidation protein products (AOPPs) in the plasma of patients with BEN, compared to the control group (p < 0.001). The urinary levels of AOPPs were higher in patients with BEN in comparison to the control (p < 0.001). The specific activity of XO was significantly lower in plasma and urine in BEN samples, compared to controls (p < 0.005). Based on these results, we hypothesize that XO might not be considered a direct systemic or local contributor to ROS production in BEN, most probably because of the diminished kidney functional tissue mass and/or AA-induced changes in purine nucleotide conformation. The increased AOPP and TBARS level in both plasma and urine in BEN may predict ROS systemic liberation with toxic local effects.

The role of lecithin cholesterol acyltransferase and organic substances from coal in the etiology of Balkan endemic nephropathy: a new hypothesis.

Balkan endemic nephropathy (BEN) occurs in Serbia, Bulgaria, Romania, Bosnia and Herzegovina, and Croatia. BEN has been characterized as a chronic, slowly progressive renal disease of unknown etiology. In this study, we examined the influence of soluble organic compounds in drinking water leached from Pliocene lignite from BEN-endemic areas on plasma lecithin-cholesterol acyltransferase (LCAT) activity. We found that changes for all samples were the most prominent for the dilution category containing 90% plasma and 10% of diluting media. Water samples from BEN villages from Serbia and Romania showed higher LCAT inhibiting activity (p=0.02) and (p=0.003), respectively, compared to deionised water and non-endemic water. A secondary LCAT deficiency could result from this inhibitory effect of the organic compounds found in endemic water supplies and provide an ethiopathogenic basis for the development of BEN in the susceptible population.

Biomarkers of oxidative damage and antioxidant enzyme activities in pre-dialysis Balkan endemic nephropathy patients.

AIM: To obtain more insight into molecular mechanisms underlying oxidative stress in Balkan endemic nephropathy (BEN), biomarkers of oxidative stress and antioxidant enzyme activities were studied in 38 pre-dialysis BEN patients, 21 healthy BEN family members and 36 healthy subjects from non-endemic areas. METHODS: Protein thiol groups (P-SH), antioxidant enzyme activities [superoxide dismutase (SOD) and glutathione peroxidase (GPX)], were determined in plasma spectrophotometrically, while malondialdehyde adducts (MDA) by enzyme immunoassay. RESULTS: BEN patients had significantly lower plasma GPX activity in comparison with values for both control groups (p = 0.016), gradually decreasing with kidney function impairment estimated by glomerular filtration rate (r = 0.53, p = 0.002). GPX activity was inversely correlated with serum urea (r = -0.627, p < 0.001), creatinine (r = -0.53, p < 0.05), urinary excretion of protein and α1-microglobulin (r = -0.44, p = 0.012; r = -0.50, p < 0.007). Significant upregulation of SOD activity was observed in healthy BEN family members (p < 0.05). While the concentration of MDA adducts was similar in all three groups, BEN patients and healthy BEN family members exhibited increased protein damage, based on fewer P-SH groups in comparison with subjects from non-BEN areas (p = 0.085; p = 0.014, respectively). CONCLUSIONS: Based on our results on increased oxidative protein damage in both pre-dialysis BEN patients and healthy BEN family members, it can be speculated that individuals from BEN areas, in general, are chronically exposed to some prooxidant environmental compounds. Moreover, decrease in plasma GPX activity, as a consequence of impaired kidney function, could further affect oxidative status in BEN patients.

Partial lecithin:cholesterol acyltransferase (LCAT) deficiency in Balkan endemic nephropathy.

The role of lipid abnormalities has been also implicated in the progression of renal diseases. The search for lipid abnormalities in Balkan endemic nephropathy (BEN) has roused sporadic interest and has not been fully elucidated. This study was performed in 54 healthy subjects from the families affected with BEN (group A), 18 members from non-affected families living in the same location (group B), and 25 control subjects (group C). Lipid profiles and lecithin:cholesterol acyltransferase (LCAT) were determined in each subject. The most striking distinction between the groups was that of the LCAT activity, which was abnormally low in group A (39 +/- 2), significantly different (P less than 0.0001) from that of the other groups. Thirty individuals from group A were those accounting for the low LCAT activity (A1). This group had a significantly lower total cholesterol and free cholesterol than all of the other subjects. The entire group A subjects had a significantly lower percentage of free cholesterol than the other two groups. There was no significant difference in HDL cholesterol between any of the groups, but group A1 had significantly higher HDL than group C (P less than 0.04). What emerges from our study is that a certain proportion of subjects from BEN families have a peculiar form of lipid abnormalities associated with an abnormal LCAT activity. At present we have no explanation for these findings. We believe that these changes may have an important role in the pathogenesis of BEN.

Erythrocyte delta-aminolevulinate dehydratase measurements in Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN) is a chronic progressive kidney disease leading to renal insufficiency. The possible etiological role of some toxic factors was considered in this study by investigating the activity of erythrocyte delta-aminolevulinate dehydratase (ALA-D), an enzyme influenced by various environmental factors. We observed that ALA-D activity was markedly decreased in patients with BEN and in 32% of their healthy family members. Glutathione concentration was found normal in all the groups studied, however, it was possible to reactivate the enzyme in vitro by addition of exogenous glutathione. Percent activation was significantly higher in the groups with decreased ALA-D activity. Blood lead levels were within normal range. The results suggest a normal synthesis of ALA-D apoenzyme, and also the presence of some factors, environmental, metabolic, or genetic, which may affect the enzyme activity through binding to the reactive groups in the active center of this enzyme or by oxidation of the reactive groups. Additional studies are necessary to further elucidate the significance of decreased ALA-D activity in BEN and their healthy relatives.

Selenium-dependent and selenium-non-dependent glutathione peroxidase in patients with Balkan endemic nephropathy.

We studied the activity of erythrocyte selenium (Se)-dependent, Se-non-dependent glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in uremic patients (UP) in clinically healthy members from families affected with Balkan nephropathy (HMF/BEN) and in healthy volunteers from endemic settlements (control group). The SOD activity was not significantly different in the groups studied and the Se-non-dependent GSH-Px activity in HMF/BEN and UP was not different from the control group. However, the activity of Se-dependent GSH-Px in UP was lower compared with the control group, whereas the mean value of the Se-dependent GSH-Px activity in HMF/BEN was not significantly different when compared with the other two investigated groups.

[The effect of duration of endemic nephropathy on serum angiotensin converting enzyme activity]. / Uticaj trajanja oboljenja na serumsku aktivnost angiotenzin konvertirajuceg enzima u pacijenata sa endemskom nefropatijom.

The effects of duration of disease on serum angiotensin converting enzyme (ACE) was measured in 60 patients with endemic nephropathy (30 men and 30 women) of age between 30 and 60 years. There were formed three groups: patients with endemic nephropathy and duration of disease less than 5 years (n = 23), patients with endemic nephropathy and duration of disease between 5-10 years (n = 17); and patients with endemic nephropathy and duration of disease 10 years and more (n = 20). The serum ACE activity was determined by the spectrophotometric method using Hip-Gly-Gly as a substrate. The activity of enzyme were expressed in units corresponding to 1 nmol of the hippuric acid that was released by the hydrolysis of Hip-Gly-Gly per minute and ml of serum. The results showed that serum ACE activity decreased in group of patients with endemic nephropathy and duration of disease 10 years and more (29.21 +/- 2.25; X +/- SEM) in comparison with group of patients with endemic nephropathy and the duration of disease less than 5 years (35.57 +/- 1.75), which was statistically significant (p < 0.03).

Serum angiotensin-converting enzyme activity in patients with endemic nephropathy.

Serum angiotensin-converting enzyme was measured in 60 patients with endemic nephropathy and in 30 healthy individuals. According to the arterial blood pressure, the patients with endemic nephropathy were further divided into groups with arterial hypertension (n = 30) and without arterial hypertension (n = 30). The activity of angiotensin-converting enzyme was determined by a spectrophotometric method using hippuryl-l-histidyl-l-leucine as a substrate. The serum activity of angiotensin-converting enzyme was significantly increased in the patients with endemic nephropathy (28.51 +/- 1.64 U/l) as compared with healthy individuals (20.83 +/- 1.33 U/l). The level of the enzyme was further increased if the endemic nephropathy was accompanied by arterial hypertension (37.09 +/- 1.45 U/l). The possible mechanisms of the increase in the angiotensin-converting enzyme activity are discussed.

Individuality in cytochrome P450 expression and its association with the nephrotoxic and carcinogenic effects of chemicals.

The susceptibility of a tissue to the toxic and/or carcinogenic effects of chemicals is determined by a variety of factors, which include their rate of metabolic activation by the cytochrome P450-dependent monooxygenases. Individual differences in the levels of cytochrome P450 expression would be expected, and are known, to give rise to profound differences in toxicological response. Such effects are almost best exemplified by the sex differences observed in the toxic effects of a variety of nephrotoxins and carcinogens. In recent work, we have shown that in species such as the mouse and rat almost all cytochrome P450 enzymes in the kidney are sexually differentiated. This difference in cytochrome P450 regulation is mediated by testosterone and explains the large differences observed in the metabolic activation, toxicity and carcinogenicity of chloroform and possibly of other compounds such as ochratoxin A. In addition to hormonal or environmental influences on cytochrome P450 expression, genetic factors have also been shown to be important. In man, this is best exemplified by the genetic polymorphism observed in the metabolism of debrisoquine and approximately 25 other drugs. This genetic defect affects approximately 5-10% of the Caucasian population and has been associated with altered susceptibility to cancer. In this presentation, the development of a simple DNA-based assay to identify affected individuals is described. Use of this assay will allow clarification of the reported association of this genetic polymorphism to susceptibility to Balkan nephropathy and cancer.

Genetic predisposition to Balkan endemic nephropathy: ability to hydroxylate debrisoquine as a host risk factor.

The objective of this study was to examine the association between efficiency of oxidative metabolism and risk for developing Balkan endemic nephropathy (BEN) and/or transitional-cell carcinoma of the urinary tract, using a case-control design controlling for age, gender and socioeconomic factors. Over 900 urine samples were taken from 646 subjects, divided into the following groups: healthy subjects from areas with no BEN; healthy subjects from villages with BEN; subjects suspected of having BEN; and subjects with BEN and/or upper urinary tract tumours (UUT). BEN patients and controls from the same villages were of similar age. The highest urinary recovery of debrisoquine was found among controls from areas with no BEN; recovery in BEN patients was only 50% of that in controls. The most interesting result is that BEN patients did not have impaired debrisoquine metabolism: subjects who metabolized less than 25% of the drug represented only 2.9% of BEN patients, 12.4% of controls from BEN villages and 12.7% of controls from outside the BEN area. The very poor metabolizers represented 1.0% of BEN patients and 4.8-5.8% of controls. the percentages of extensive metabolizers in the same groups were 86.3, 64.5 and 67.4%, respectively. The mean metabolic ratio rose progressively from BEN patients less than suspected BEN patients less than controls from BEN villages less than controls from non-BEN villages; the maximum metabolic ratios were 40, 51, 72 and 87, respectively. The cumulative distribution of the 8-h urinary debrisoquine metabolic ratios, presented as a normal probability plot, formed a discrete population with values over 10. The distribution among patients with BEN/UUT indicates a predominance of extensive debrisoquine hydroxylation and a lack of poor metabolizers. These results are consistent with the hypothesis that the efficiency of oxidative metabolism is greater in BEN patients and that it may be one of the key host factors determining predisposition to these diseases.