Marcel Castegnaro

Balkan endemic nephropathy and associated urinary tract tumours: a review on aetiological causes and the potential role of mycotoxins

A series of publications in the 1950s described a kidney disease in Bulgaria, the former Yugoslavia and Romania that became known as Balkan endemic nephropathy (BEN). The disease was qualified by World Health Organisation (WHO) experts as ‘progressive and very gradually developing renal failure with insidious onset … The last stage shows marked fibrosis …’. BEN is characterized by tubular degeneration, interstitial fibrosis and hyalinization of glomeruli accompanied by enzymuria and impaired renal function without nephrotic syndrome. Later, an association between BEN and tumours of the kidney pelvis and ureter was recognized, so that the problem of BEN became not only nephrological, but also oncological. There may also be an association with increased urinary bladder cancer incidence, although many confounding factors may interfere in the analysis of data for this organ. In view of the very intimate association between BEN and the urinary tract tumours (UTT), the term ‘endemic uropathy’ has been proposed. Several hypothesis concerning the aetiology of these diseases has been investigated, which include: predisposing genes factors, environmental factors (heavy metals, minerals, bacteria, leptospira, viruses, fungal toxins and, most recently, pliocene lignites). This paper reviews the different hypotheses about the aetiology of endemic uropathy and pays particular attention to the role of fungal toxins.

Ochratoxin A in human blood in relation to Balkan endemic nephropathy and urinary system tumours in Bulgaria

Ochratoxin A is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy and the associated urinary tract tumours. Contamination of cereals by this mycotoxin has been found to be more frequent in areas of endemic nephropathy than in areas where the disease is absent. As ochratoxin A binds to serum albumin, it should be detectable in biological fluids from exposed populations. A survey was thus conducted to determine the occurrence of ochratoxin A in blood from people living in the endemic area who were either affected or unaffected by the two diseases and in blood from people living in control regions where these diseases do not occur. Blood samples were collected in 1984, 1986, 1989 and 1990. Ochratoxin A was found more frequently and at higher levels in blood from patients with Balkan endemic nephropathy and/or urinary tract tumours than in blood from unaffected people from endemic and control areas. These findings suggest further that ochratoxin A is involved in the etiology of the two diseases.

Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with DNA adduction

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, has been implicated as an etiologic agent in the Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Compared with unaffected individuals, patients suffering from BEN and/or urinary tract tumors were more frequently found to have a capacity for rapid debrisoquine (DB) metabolism, a metabolic reaction related mostly to cytochrome P450 (CYP) 2D in humans. Earlier studies, using female DA and Lewis rats phenotyped as poor or extensive DB metabolizers respectively, revealed a parallelism between DB‐4 hydroxylation and OTA‐4 hydroxylation. To investigate whether genetic polymorphism modifies tumor induction, we have compared both OTA‐induced renal carcinogenicity and DNA adducts in DA and Lewis rats of both sexes. OTA induced renal adenocarcinoma, DA male rats being most responsive, while DA females were resistant. Lewis rats showed an intermediate renal tumor response. OTA also induced malignant transitional cell carcinomas of the bladder in DA male rats only. DNA adducts in the kidney, as judged by the nature of spots and prevalence in OTA‐treated animals, were significantly correlated with renal carcinogenicity of OTA, being highest in DA males and lowest in DA females. A parallelism between karyomegalies and tumors of the kidney was observed. In conclusion, our results classify OTA as a genotoxic carcinogen in rats, with sex‐specific response controlled in part by drug‐metabolizing enzymes that convert OTA into reactive intermediates. Int. J. Cancer 77:70–75, 1998.© 1998 Wiley‐Liss, Inc.

Sex- and strain-specific expression of cytochrome P450s in Ochratoxin A-induced genotoxicity and carcinogenicity in rats

Ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin, is implicated in the etiology of Balkan endemic nephropathy (BEN), a chronic disease affecting populations in the Balkans. Patients suffering from Balkan endemic nephropathy, urinary‐tract tumors, or both are more frequently extensive metabolizers of debrisoquine than persons unaffected by these conditions. As shown previously (Castegnaro et al., Int J Cancer, 77:70–75, 1998), OTA induction of renal tumors is markedly sex‐ and strain‐specific in Dark Agouti (DA) and Lewis rats, with DA males being most responsive and DA females being resistant; only DA females were phenotyped as slow debrisoquine metabolizers. Formation of OTA‐related DNA adducts in the kidney was closely correlated with renal carcinogenicity. To elucidate the critical biotransformation enzymes involved in OTA genotoxicity and carcinogenicity, the expression of cytochrome P450s (CYPs) 1A, 2A, 2B, 2C, 2D, and 3A in the kidney and liver microsomes of untreated and OTA‐treated DA and Lewis rats (both sexes) was determined by western blot analysis. Chronic OTA treatment was found to modify CYP expression in kidney and liver. In the animals most susceptible to renal OTA carcinogenicity and DNA adduct formation, the OTA‐toxifying isoforms (CYPs 2C11, 1A2, and 3A) were highly expressed in the liver, and little of the OTA‐detoxifying isoforms (CYPs 1A1 and 2A) was detected. CYP2D was not expressed in DA rats and therefore is not involved in these phenomena. Our results confirm that the strain‐ and sex‐specific genotoxic response of OTA is controlled, in part, by CYP‐mediated metabolic reactions that convert OTA into DNA‐reactive intermediates. Mol. Carcinog. 23:76–85, 1998.

Genotoxic activity of potassium permanganate in acidic solutions

Potassium permanganate (KMnO4) combined with sulfuric acid is a strongly oxidizing mixture which has been recommended for the destruction and the decontamination of various mutagens/carcinogens in the publication series of the International Agency for Research on Cancer. Evaluation of the genotoxicity of 4 potassium permanganate solutions was performed using a microtechnique of the Ames test with the tester strains TA97, TA98, TA100 and TA102 with and without metabolic activation. Presence of direct-acting mutagens was detected in all the samples with the tester strain TA102 without S9 mix (163-357 revertants/microliters of the solutions). Three samples containing either acetone or ethanol as an organic solvent also induced a mutagenic response on tester strain TA100 without S9 mix (167-337 revertants/microliters). In addition, DNA damage in human peripheral blood lymphocytes was also measured for one of the mixtures by a new technique: the single-cell gel assay (SCGA). A sample with no organic solvent induced DNA damage in human lymphocytes with a dose-response relationship as determined by SCGA. The major mutagenic agent generated by the permanganate solutions was found to be manganese ion (Mn2+). Both manganese sulfate (MnSO4) and manganese chloride (MnCl2) gave mutagenic dose-response relationships on tester strain TA102 without S9 mix. The mutagenic potencies were 2.8 and 2.4 revertant/nmole for MnSO4 and MnCl2 respectively. MnCl2 also induced DNA damage in human lymphocytes as determined by the SCGA. The genotoxic effects of KMnO4 in acidic conditions were probably mediated by the conversion of MnO4- to Mn2+. KMnO4 in alkaline solutions did not produce mutagenic species and may offer an alternative for the degradation of genotoxic compounds.

Formation of ochratoxin a metabolites and DNA-adducts in monkey kidney cells

Monkey kidney cells (named Vero cells) were incubated with increasing doses of ochratoxin A (10-100 microM). The inhibiting concentration 50% (IC50) on protein synthesis was about 14 microM in the presence of 5% fetal calf serum and 37 microM in the presence of 10% fetal calf serum. Some metabolites of ochratoxin A, including the chlorinated dihydroisocoumarin moiety of OTA (OT alpha), 4-[S]-hydroxy-OTA and 4-[R]-hydroxy-OTA were detected by HPLC in the mixture of cell homogenate after a 24 h incubation with 10 and 25 microM of OTA. Using the 32P-postlabelling method, several DNA-adducts, similar to those formed in mouse kidney after OTA treatment, were detected in monkey kidney cells. Thus, Vero cells are suitable for genotoxic and cytotoxic studies in relation to the metabolism of nephrotoxic xenobiotics such as OTA.

Ochratoxin A contamination of cereals in an area of high incidence of Balkan endemic nephropathy in Bulgaria.

In an effort to provide further evidence for the hypothesis that a mycotoxin is involved in the aetiology of Balkan endemic nephropathy and that the latter is associated with the occurrence of urinary system tumours, a survey was made of ochratoxin A contamination of cereal samples from an area of Bulgaria where both endemic nephropathy and urinary system tumours are prevalent and from non-endemic areas. In all, 130 samples of beans, maize and wheat flour were analysed. Ochratoxin A levels were 16.7% (25-27 micrograms/kg) in bean samples from the endemic area and 7.1% (25-50 micrograms/kg) in those from the control areas: in maize samples, the levels were 27.3% (25-35 micrograms/kg) and 9.0% (10-25 micrograms/kg), respectively.

Evaluation of the genotoxic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay

The genotoxicity of metronidazole (MZ) and dimetridazole (DZ) has been evaluated in human lymphocytes using the comet assay. The test has been performed using 3 doses (58.4, 175.2 and 292.1 microM for MZ; and 70.9, 212.6 and 354.3 microM for DZ) under 3 experimental protocols: aerobiosis, anaerobiosis (90% N2, 10% CO2) and with the presence of the microsomal fraction S9 mix. The effects of 4 antioxidants (8-hydroxyquinoline (8HQ), vitamin C (VitC), catalase (CAT) and superoxide dismutase (SOD), have been investigated on DNA damage generated by fixed concentrations of MZ (292.1 microM) and DZ (354.4 microM). In aerobic conditions, MZ and DZ produced significant dose-response relationships. The dose-related effects of both drugs decreased or were abolished in anaerobic conditions or in presence of S9 mix. 8HQ, VitC, CAT and SOD induced dose-related protective responses against DNA damage due to MZ and DZ. These findings suggest that MZ and DZ induce DNA damage in human lymphocytes through the futile cycle. The one-electron reduction of the drugs leads to the production of nitro radical anions. In the presence of oxygen, these radicals are reoxidized and generate oxygen-activated species.

Analytical method for the determination of sphinganine and sphingosine in serum as a potential biomarker for fumonisin exposure

The toxins produced by Fusarium moniliforme, which include fumonisins, are possible human carcinogens. Fumonisins are inhibitors of de novo sphingolipid biosynthesis. Alterations of the ratio of sphinganine (Sa) to sphingosine (So) in urine and serum has been proposed as a possible biomarker of exposure to this toxin. A new method was developed for their analysis in tissues and urine. This work describes the further adaptation of the method to the analysis of Sa and So in serum and its validation in sera of untreated and fumonisin B1 (FB1 ) treated rats and mice. No significant differences in the Sa/So ratios were observed in the FB1 treated rats. In mice, the increase was only of marginal statistical significance. Determination of Sa/So ratios in human sera could readily be made in small volumes (from 0.3 to 0.5 ml) of serum.

Influence of ethyl alcohol on carcinogenesis with N-nitrosodimethylamine

This paper presents the results of an experiment on the combined action of nitrosodimethylamine and ethyl alcohol in C57BL mice. As shown, alcohol can act to change the target organ of that liver carcinogen by favouring development of olfactory neuroepitheliomas, which infiltrate the frontal lobe of the brain.