DNA ploidy distribution in renal tumours induced in male rats by dietary ochratoxin A

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Abstract

DNA ploidy distribution, measured in experimental renal tumours that occurred in twelve ageing male Fischer rats derived from carcinogenicity experiments on ochratoxin A (OTA) in response to chronic dietary exposure, was diploid in all renal adenomas and aneuploid in all carcinomas, correlating with their typical organised and disorganised histopathology, respectively. Aneuploidy was also detected in renal tissue in which karyomegaly, induced by OTA, was analogous to that caused by the fungus Penicillium polonicum. Thus, the experimental rat renal carcinoma could arise within an adenoma directly from certain persistent karyomegalic tubular epithelial cells long after their particular genetic damage has been caused during a protracted period of OTA insult.

Introduction

Ochratoxin A (OTA) is well known to have diverse toxicological effects (nephrotoxic, immunotoxic, teratogenic, carcinogenic) on animals. However, the aspect of current concern is the validity for human risk assessment of the rat model in which the kidney is a specific target organ for induction of tumours. These are typically discovered, mainly in male rats, during the last quarter of a standard 2-year lifetime period (Boorman, 1989). Recently a wide range of experimental rat renal tumours, derived by exposure to diet contaminated artificially with OTA and studied histopathologically, have become available and to which has been applied a modern computerised procedure for measurement of DNA ploidy distribution in nuclei isolated from the standard wax-embedded tissues.

Application of findings from a rat renal cancer model to human renal cell carcinoma (RCC) might be easy if OTA could fit plausibly into a well-defined array of causal factors in the human disease. However, RCC is still mostly idiopathic; only about 2% of cases are of a familial type with a known genetic basis (Maher, 2004). Nevertheless, from the extensive medical literature, two recent examples illustrate increasing understanding of the important evolving tumour dynamics that determines whether distant metastasis is likely to have occurred before surgical intervention. Flow cytometry analytical data (Pinto et al., 2005) of 64 RCC obtained by radical nephrectomy during 1991–1996 in Portugal, showed that half were DNA aneuploid. Ploidy correlated significantly with pathological staging which also, at stages 3 and 4, correlated with worse disease-related survival. Considering outcome, aneuploid tumours were estimated to predict a 15-fold higher risk of poor prognosis than diploid tumours. Consequently, one or more foci of DNA aneuploidy arising in a diploid RCC is now recognised as a source of the distant aneuploid metastases that predict poor prognosis. Genetic change in aneuploid foci in RCC, additional to that already in place within the main mass of the tumour, could be expected and would generate the more aggressive metastatic tissues. Improving screening efficiency for such foci is the basis for recommending analysis of five regions of tumour specimens from surgical resection of a large tumour mass to amplify pathological grading (Li et al., 2005).

Another study (Li et al., 2002) on 67 consecutive French RCC patients (1998–2002), analysed five regions (non-necrotic) of each tumour and showed relationship between aneuploidy and tumour size according to TNM 1997 classification. Tumours were further categorised either as diploid or as either heterogeneously or homogeneously aneuploid. A plot of analytical data for aneuploid tumours showed a continuum, from diploid to tetraploid, within which the hypertriploid clone was the most abundant. Hypertriploidisation was considered to be a major pathway towards the aneuploidisation of RCC.

Thus the primary hypothesis for the present study is that there may be a distinction between the DNA diploidy expected in small adenomas arising in rat renal parenchyma in response to OTA and the ploidy distribution in the disorganised renal carcinomas that appear to have arisen subsequently from adenomas. In this context it was also important to quantify the ploidy distribution of the karyomegaly induced by OTA long before neoplasms arise. Measurement of DNA ploidy has apparently not previously been performed on renal tumours from experimental animals and thus, within the limited available resources, tumours from several current experiments were included in the present study.

Section snippets

Production of diets contaminated with OTA

Production of OTA was achieved by growing Aspergillus ochraceus isolate D2306 (Harris and Mantle, 2001) in shaken solid substrate fermentations at 28 °C for 2 weeks to yield batches of a product containing 5–6 mg OTA/g. Specifically, 40 g of sterilised shredded wheat (Cereal Partners UK, Welwyn Garden City, UK) in 500 ml Erlenmeyer flasks was inoculated with a concentrated spore suspension in water (16 ml) and the flasks shaken at 200 rpm and 10 cm eccentric throw. An aliquot of each fermentation

Results

Tumours and associated tissues and the analytical results are described and illustrated in Table 1 and Fig. 1, Fig. 2. Classification of tumours as adenoma or carcinoma was based primarily on histopathological criteria (Eble et al., 2004). Adenomas used for the present study were mostly small compression tumours of varying size, bounded by a stretched layer of renal cortex and bounded by the capsule. The tumours thus maintained a roughly spherical neoplasm. Internally, organised arrangement of

Discussion

The analytical findings have substantiated a marked change in DNA ploidy during the adenoma-carcinoma continuum in OTA-generated rat renal tumours. The consistent correlation of DNA aneuploidy with the proliferative renal carcinomas, contrasting with the diploidy of adenomas, ranged across all the groups of rat renal tumours arising during experiments in the recent EU-funded programme (QLKI-CT-2001-011614) and that were studied and archived as wax-embedded preparations.

Aneuploid carcinomas in

Acknowledgements

Experimental tissue was generated during participation in Project No. QLK1-2001-01614 of the 5th Framework Program of the European Union. Histological preparations were made in the Breast Pathology Unit, Guy's Hospital, London, where photomicrography equipment was used and where helpful discussions with Dr. Corrado D’Arrigo led to the present collaboration. We are grateful to Guy's and St Thomas’ Charity for research funding to Prof. E. Odell.

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