Co-Exposure to Aristolochic Acids I and II Increases DNA Adduct Formation Responsible for Aristolochic Acid I-Mediated Carcinogenicity in Rats

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804–2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

MO374DISCLOSURE OF THE RESULTS OF A 40-YEARS RESEARCH OF UPPER TRACT UROTHELILAL CANCER AND BALKAN ENDEMIC NEPHROPATHY

Background and Aims The general significance of Balkan endemic nephropathy (BEN) is the association with upper tract urtohelial cancer (UTUC). In published papers studying these two entities, there is large difference between the obtained results. By UTUC research, obtained results are the most diverse in relation to the period and region of research. The aim of the research is to show the discrepancy between the results of the research of UTUC of Jablanica district in relation to the observation period and the type of settlement. Method The research period lasted from 1978-2017. During the analysis of the frequency of UTUC, we used the operative material of Urology Department, Health Care Center, Leskovac , and Urology Clinic, Clinical Center, Nis, Clinical Center, Nis. For practical reasons, this period was devided into two periods, the first (1978-1997) and the second (1998-2017). In order to make classification of settlements we used the data of the Institute of Nephrology and Hemodialysis in Nis (A-endemic regions, B-hypo-endemic, C-non-endemic urban, D-non-endemic rural regions). Data on the total number of Jablanica region population were obtained on the basis of the censuses from 1991 and 2011. The average annual incidence rate (AAIR) was calculated per 100 000 people. Finally, we jointly observed groups A and B (endemic areas) C and D (non-endemic areas) for UTUC. Results The average annual incidence rate (AAIR) in the period of 1978-2017 in endemic settlements of Jablanica region was (11.82), while in hypo-endemic was (4.49) and non-endemic (0.83). The data demonstrated that inhabitants of endemic settlements has 14.24 times higher UTUC frequency in comparison to non-endemic settlements in time span of research. Our research in Jablanica region also demonstrated unexpectedly high frequency of on UTUC not only in endemic settlements with BEN (Kutles village- 1 tumor per 99.63 and AAIR 40.15), but also in some of the non-endemic area (Brejanovac village-1 tumour per 98.75 people and AAIR of 40.50; Rudare village-1 tumour per 139.50 people and AAIR of 28.67; Bogojevce village-1 tumour per 187.63 people and AAIR 21.32). This occurrence of UTUC frequency in some non-endemic settlements refers to the first observed period until no occurrence was recorded in the second observed period. There is a higher UTUC frequency in endemic settlements of 11.37 in the first period (A- AAIR 21.95), while in hypo-endemic (B-AAIR 11.82) is 3.64 higher incedence. In non-endemic settlements (C,D-AAIR 1.09) there is 1.63 higher incidence in comparison to the second period. Observing the periods, there is a higher UTUC frequency of five times in endemic settlements (A, B) of Jablanica region in the first period comparing to the second. The linear trend of UTUC in the 40-year period demonstrates a slow decrease (y= -0.0797x + 4.2846; r2 = 0.2028) in Jablanica region. In the same observed period, linear trend of BEN is in corelation of decreased linear trend of UTUC (y= -0.164x+6.0669; r2 =0.748). Conclusion A forty-year study of UTUC in the Jablanica region showed a discrepancy between the results in relation to the observation period and the type of settlement, which coincides with the generally accepted view that epidemiology is the most fascinating part of BEN. In endemic settlements, in the second observed period, the frequency of UTUC was registered five times lower than in the first, which coincides with the decrease in the frequency of BEN in these settlements.

Rat Tumour Histopathology Associated with Experimental Chronic Dietary Exposure to Ochratoxin A in Prediction of the Mycotoxin’s Risk for Human Cancers

Mammalian animal toxicity of ochratoxin A (OTA) has focused largely in the past half-century on pigs because of initial recognition of it as a principal cause of intermittent growth suppression and renal disease caused by mouldy feed. Subsequent classical toxicology has used laboratory rodents because renal pathology in pigs raised questions concerning possible involvement in the human idiopathic bilateral renal atrophy of Balkan endemic nephropathy for which OTA was a focus of attention for human nephropathy through 1980s and into 2000s. Emphasis on human nephropathy has more recently concerned the plant metabolite aristolochic acid. Recognition that agricultural management can often minimise food and feed-stuff spoilage by OTA-producing Aspergilli and Penicillia has moderated some of the risks for animals. Legislation for human food safety combined with sophisticated analysis generally provides safety in the developed world. Chronic experimental exposure of male rats, in the absence of clinical dis-ease, specifically causes renal cancer. The possibility of this as a unique model for the human has generated considerable experimental evidence which may be more directly relevant for carcinogenesis in the complex kidney than that obtained from biochemical toxicities in vitro. Nevertheless, there does not appear to be any case of human renal or urinary tract cancer for which there is verified etiological proof for causation by OTA, contrary to much claim in the literature. To contribute to such debate, histopathology review of OTA/rat renal cancers, augmented where appropriate by immune profiles, has been completed for all remaining tumours in our research archive. Overall consistency of positivity for vimentin, is matched with occasional positives either for CD10 or the cytokeratin MNF 116. The current situation is discussed. Suggestion that OTA could cause human testicular cancer has also been challenged as unsupported by any experimental findings in rats, where the Leydig cell tumour immune profile does not match that of human germ cell neoplasms.

Simultaneous ipsilateral rhabdoid renal cell carcinoma and multifocal urothelial carcinoma of the ureter in a patient from the region of Balkan endemic nephropathy: Case report and literature review

Introduction. Simultaneous ipsilateral coexistence of renal cell carcinoma (RCC) and upper urinary tract urothelial carcinoma (UTUC) rarely occurs. Balkan endemic nephropathy (BEN) is a chronic degenerative tubulointerstitial renal disease, strongly associated with UTUC. Case outline. ? 60-year-old man from the region of BEN was referred to our clinic due to right flank pain, fever, and purulent discharge from the cutaneous fistulous opening in the right lumbar area. Multislice computed tomography urography scan showed right-side pyonephrosis and nephrocutaneous fistulous tract between the kidney and the skin in the right lumbar region. Cystoscopy detected papillary tumor protruding from the right ureteric orifice. Right-side nephroureterectomy with bladder cuff excision (BCE) was performed. Histopathological examination revealed rhabdoid RCC of the kidney and multifocal urothelial carcinoma of the ureter. Conclusion. Our case report and literature review indicate that due to rising incidence of multiple primary malignant neoplasms, when treating patients with RCC or UTUC, and especially those from the region of BEN, one should keep in mind the likelihood of synchronous or metachronous occurrence of these tumors.

Predictive Markers for Malignant Urothelial Transformation in Balkan Endemic Nephropathy: A Case–Control Study

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease frequently accompanied by urothelial carcinoma (UC). In light of the increased UC incidence and the markers observed in BEN patients with developed UC, the aim of the current case–control study is to assess survivin, p53 protein, growth factors and receptors (VEGF, VEGFR1, IGF I, IGF-1R and IGFBP5), tumor marker (TF)/CD142, circulating soluble Fas receptor and neopterin, as potentially predictive markers for UC in patients with BEN (52 patients), compared to healthy, age-matched subjects (40). A threefold increase was registered in both circulating and urinary survivin level in BEN patients. Especially noticeable was the ratio of U survivin/U Cr level five times the ratio of BEN patients associated with standard renal markers in multivariate regression models. The concentrations of VEGF, VEGFR1, (TF)/CD142, (sFas) were not significantly different in BEN patients, while urinary/plasma level demonstrated a significant decrease for VEGF. The levels of IGF I, IGFBP5 and IGF-1R were significantly reduced in the urine of BEN patients. Plasma concentration of neopterin was significantly higher, while urinary neopterin value was significantly lower in BEN patients compared to healthy controls, which reflected a significantly lower urine/plasma ratio and low local predictive value. As BEN is a slow-progressing chronic kidney disease, early detection of survivin may be proposed as potential predictor for malignant alteration and screening tool in BEN patients without the diagnosis of UC.

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.