A new biological assay, utilizing parasitic protozoa, for screening carcinogenic substances which induce bladder cancer in man and other mammals

Injections of aromatic amines (β-naphthylamine, benzidine, O-dianisidine or N-2-fluorenyl acetamide), tryptophan metabolites (3-hydroxyanthranilic acid, xanthurenic acid or LD-kynurenine sulphate), oestrone, and nicotine, which are known bladder carcinogens in man and some other mammals induced sexual reproduction (encystation) in Opalina sudafricana when injected into its host Bufo regularis. This may be used as a new biological assay for screening substances which induce bladder cancer in man and some other mammals. It is speculated that the metabolites of the injected carcinogenic substances used in this work are excreted in the urine of the host, hydrolysed by the hydrolytic enzymes and become carcinogenic. These carcinogenic metabolites reach the parasites in the rectum of the toads and induce them to divide mitotically to form small forms which eventually encyst. It is speculated that the presence of cysts in the rectum of the injected toads is indicative that a carcinogenic effect took place in the parasites. Oestrone is the only carcinogenic substance which induced encystation in the opalinids in vitro. Urine of toads injected with β-naphthylamine, benzidine, O-dianisidine, N-2-fluorenyl acetamide, 3-hydroxyanthranilic acid, xanthurenic acid, DL-kynurenine sulphate, oestrone and nicotine induced cyst formation in the parasites in vitro.     

Evaluation of a new model to detect bladder carcinogens or co-carcinogens; results obtained with saccharin, cyclamate and cyclophosphamide.

A sensitive rat model has been designed to detect potential weak bladder carcinogens or co-carcinogens. The test compound is given to animals which have received a single initiating, but non-carcinogenic, dose of N-methyl-N-nitrosourea (MNU). The model has been used to investigate two compounds currently under suspicion as weak bladder carcinogens, namely sodium saccharin and sodium cyclamate, and one compound known to be cytotoxic but not carcinogenic for the bladder epithelium namely cyclophosphamide. For comparison, these three compounds were also tested as solitary carcinogens in animals not pre-treated with MNU. At the very high dose levels used, sodium saccharin and sodium cyclamate were weak solitary carcinogens producing 4/253 and 3/228 bladder tumours respectively, and the first of these tumours did not appear for more than 80 weeks. When tested in the MNU/rat model more than half the animals receiving either sodium saccharin or sodium cyclamate developed bladder tumours from 10 weeks onwards. By contrast, cyclophosphamide failed to produce any tumours when tested either as a solitary carcinogen or in the MNU/rat model. It must be emphasized that the doses of saccharin and cyclamate used were far higher than those consumed by man, including diabetics, and these results should not be directly extrapolated to man without careful consideration of many other factors including negative epidemiological findings. The theoretical basis of the model is discussed and also the relevance, in terms of environmental human exposure, of detecting compounds which have a synergistic effect with other known bladder carcinogens. It appears that this model can be used to detect a carcinogenic or co-carcinogenic potential in compounds which are organotropic for the bladder more rapidly and with fewer animals than if the compounds are tested as solitary carcinogens by more conventional methods. It is suggested that it could be used to detect those compounds which require further investigation.     

Banding carcinogenic risks in developed countries: A procedural basis for qualitative assessment

Readily achieved comparative assessment of carcinogenic risks consequent upon environmental exposures may increase understanding and contribute to cancer prevention. Procedures for hazard identification and quantitative risk assessment are established, but limited when addressing novel exposures to previously known carcinogens or any exposure to agents having only suspected carcinogenic activity. To complement other means of data evaluation, a procedure for qualitative assessment of carcinogenic risk is described. This involves categorizing the relevant carcinogen and circumstances under which exposure occurs. The categories for carcinogens are those used for hazard identification and involve whether the agent is (1) a recognized carcinogen for humans; (2) probably or (3) possibly carcinogenic for humans; (4) characterized by inadequate evidence of carcinogenicity; or (5) lacking carcinogenicity. Exposure is categorized by whether it is one which (1) establishes the agent as a recognized carcinogen; (2) is taken into account in establishing carcinogenicity status; (3) is distinct from those providing clearest evidence of carcinogenicity; (4) is not characterized in relation to carcinogenicity; or (5) involves an exposure in which absence of carcinogenic outcome is observed. These two categories of evidence allow the risk inherent in a situation to be banded as indicative of a proven, likely, inferred, unknown or unlikely carcinogenic outcome, and further characterized using sub-bands. The procedure has been applied to about fifty situations. For recognized carcinogens, including asbestos and polycyclic aromatic hydrocarbons, risks consequent upon occupational exposure, the impact of point source pollution, residence near contaminated sites and general environmental exposure are allocated across the proven band and a likely sub-band. For solvents, pesticides and other compounds having less clearly established carcinogenicity, impact on residents living near a production site, or near earlier related industrial activity is allocated to certain inferred sub-bands. Unknown carcinogenic outcome, which identifies exposure to an agent with inadequate evidence of carcinogenicity rather than being indicative of equivocal or negative data in any context, indicates both the impact of certain pollutants and user-exposure to some consumer products. Situations allocated to the unlikely risk band principally involve certain consumer products. Overall, such risk assessment may be of greatest worth in focusing community attention on proven causes of cancer and associated preventive measures.     

Evaluation and Spatial Diffusion of Health Risk of Persistent Organic Pollutants (POPs) in Soils Surrounding Chemical Industrial Parks in China

A case study of the cancer risk to humans posed by persistent organic pollutants (POPs) in an industrial area of China, which has a long history of contamination from many sources, is presented. Relatively great concentrations of POPs around the chemical industrial parks have the potential to be chronically carcinogenic to local people. Sixteen individual PAHs listed for priory control by the U.S. Environmental Protection Agency (USEPA), metabolites of DDTs, and isomers of HCHs were measured in soils and a human health risk assessment was conducted by use of USEPA exposure models for children and adults, respectively. Geostatistical methods were used to simulate the spatial diffusion of potential carcinogenic risk, and non-parametric Mann-Whitney U and Kruskal-Wallis tests were employed to analyze the impact of point sources on the surrounding area. The mean value of the sum of Excess Lifetime Cancer Risk (∑ELCR) exceeded the generally acceptable risk level of 1.0E-06 recommended by the USEPA for carcinogenic chemicals. The maximum ∑ELCR was 2.9E-04 for children, which was observed inside the chemical industrial parks. Contamination at the chemical industrial parks caused significant spatial diffusion of ELCR values caused by PAHs, DDT, and HCH.     

Infection with the carcinogenic human liver fluke, Opisthorchis viverrini

Throughout Southeast Asia there is a strikingly high incidence of cholangiocarcinoma (CCA--hepatic cancer of the bile duct epithelium), particularly in people from rural settings in Laos and Northeast Thailand who are infected with the liver fluke, Opisthorchis viverrini, one of only three carcinogenic eukaryotic pathogens. More ubiquitous carcinogenic microbes, such as Helicobacter pylori, induce cancer in less than 1% of infected people, while as many as one-sixth of people with opisthorchiasis will develop CCA. The mechanisms by which O. viverrini causes cancer are multi-factorial, involving mechanical irritation from the activities and movements of the flukes, immunopathology, dietary nitrosamines and the secretion of parasite proteins that promote a tumourigenic environment. Genomic and proteomic studies of the liver fluke secretome have accelerated the discovery of parasite proteins with known/potential roles in pathogenesis and tumourigenesis, establishing a framework towards understanding, and ultimately preventing, the morbidity and mortality attributed to this highly carcinogenic parasite.     

Parasites and malignancies, a review, with emphasis on digestive cancer induced by Cryptosporidium parvum (Alveolata: Apicomplexa)

The International Agency for Research on Cancer (IARC) identifies ten infectious agents (viruses, bacteria, parasites) able to induce cancer disease in humans. Among parasites, a carcinogenic role is currently recognized to the digenetic trematodes Schistosoma haematobium, leading to bladder cancer, and to Clonorchis sinensis or Opisthorchis viverrini, which cause cholangiocarcinoma. Furthermore, several reports suspected the potential association of other parasitic infections (due to Protozoan or Metazoan parasites) with the development of neoplastic changes in the host tissues. The present work shortly reviewed available data on the involvement of parasites in neoplastic processes in humans or animals, and especially focused on the carcinogenic power of Cryptosporidium parvum infection. On the whole, infection seems to play a crucial role in the etiology of cancer.     

Genetic Mechanisms and Aberrant Gene Expression during the Development of Gastric Intestinal Metaplasia and Adenocarcinoma

Gastric adenocarcinoma occurs via a sequence of molecular events known as the Correa’s Cascade which often progresses over many years. Gastritis, typically caused by infection with the bacterium H. pylori, is the first step of the cascade that results in gastric cancer; however, not all cases of gastritis progress along this carcinogenic route. Despite recent antibiotic intervention of H. pylori infections, gastric adenocarcinoma remains the second most common cause of cancer deaths worldwide. Intestinal metaplasia is the next step along the carcinogenic sequence after gastritis and is considered to be a precursor lesion for gastric cancer; however, not all patients with intestinal metaplasia develop adenocarcinoma and little is known about the molecular and genetic events that trigger the progression of intestinal metaplasia into adenocarcinoma. This review aims to highlight the progress to date in the genetic events involved in intestinal-type gastric adenocarcinoma and its precursor lesion, intestinal metaplasia. The use of technologies such as whole genome microarray analysis, immunohistochemical analysis and DNA methylation analysis has allowed an insight into some of the events which occur in intestinal metaplasia and may be involved in carcinogenesis. There is still much that is yet to be discovered surrounding the development of this lesion and how, in many cases, it develops into a state of malignancy.Key Words: Intestinal metaplasia, gastric cancer, aberrant gene expression, genetic markers.     

CYP4B1 is a possible risk factor for bladder cancer in humans

In experimental animals such as rats and rabbits, CYP4B1 has an important role in mutagenic activation of procarcinogens in bladders. In human bladders, it is not clear whether CYP4B1 has such role or not. In the present study, human bladder microsomes activated 2-aminofluorene which is a typical substrate for CYP4B1 and is a bladder carcinogen. CYP4B1 was detected in the human bladder microsomes by immunoblotting. Furthermore, we developed a microassay for CYP4B1 mRNA by performing real-time RT-PCR. Using this method, CYP4B1 mRNA levels were assayed in transurethal resection samples from the bladders of patients with bladder tumors. The bladder-tumor patients had a significantly higher expression of CYP4B1 than the nonbladder tumor patients. These findings suggest that a high expression of CYP4B1 increases the risk of bladder tumor by activation of carcinogenic aromatic amines. This approach could be an important tool in the assessment of human bladder cancer risk.     

Qualitative and quantitative procedures for health risk assessment.

Numerous reactive mutagenic electrophiles are present in the environment or are formed in the human body through metabolizing processes. Those electrophiles can directly react with DNA and are considered to be ultimate carcinogens. In the past decades more than 200 in vitro and in vivo genotoxic tests have been described to identify, monitor and characterize the exposure of humans to such agents. When the responses of such genotoxic tests are quantified by a weight-of-evidence analysis, it is found that the intrinsic potency of electrophiles being mutagens does not differ much for the majority of the agents studied. Considering the fact that under normal environmental circumstances human are exposed to low concentration of about a million electrophiles, the relation between exposure to such agents and adverse health effects (e.g., cancer) will become a 'Pandora's box'. For quantitative risk assessment it will be necessary not only to detect whether the agent is genotoxic, but also understand the mechanism of interaction of the agent with the DNA in target cells needs to be taken into account. Examples are given for a limited group of important environmental and carcinogenic agents for which such an approach is feasible. The groups identified are agents that form cross-links with DNA or are mono-alkylating agents that react with base-moieties in the DNA strands. Quantitative hazard ranking of the mutagenic potency of these groups of chemical can be performed and there is ample evidence that such a ranking corresponds with the individual carcinogenic potency of those agents in rodents. Still, in practice, with the exception of certain occupational or accidental exposure situations, these approaches have not be successful in preventing cancer death in the human population. However, this is not only due to the described 'Pandora's box' situation. At least three other factors are described. Firstly, in the industrial world the medical treatment of cancer in patients occurs with high levels of extremely mutagenic agents. Actually, both in number of persons and in exposure levels such medical treatment is the single largest exposure of humans to known carcinogens. Although such treatments are very effective in curing the tumor as present in the patient, the recurrence of cancer in those patients later in life is very high. In other words: "curing cancer is not the same as preventing cancer death in the human population". Secondly, the rate of cancer death in the human population is also determined by the efficacy in which other major causes of death are prevented. For instance, cardiovascular diseases are the major cause of death in humans in the industrialized world. There is evidence that the treatment of cardiovascular diseases is more successful than that of cancer. On a population level this will result in increase of cancer being the ultimate death cause. Finally, the improvement of medical treatment of diseases together with an improved quality of life will lead to increase average age of the population. Because the onset of most cancer is long after the exposure to carcinogens-in human often more than 30 years-cancer is predominantly a disease of the old age. This means that if the average age of human increases, there will be a selective preference of cancer becoming an even more important cause of death. This especially will be pronounced in those countries were the age distribution in a population is abnormal.     

Evidence for a role of prostaglandins in atropine-resistant transmission in the mammalian urinary bladder

It has been known for many years that atropine and other anti-cholinergic drugs readily inhibit the contractile responses of the detrusor portion of the mammalian urinary bladder to exogenous acetylcholine, but only partially inhibit responses to nerve or transmural stimulation (1,2). These findings suggest that the excitatory innervation to the bladder consists not only of cholinergic nerves but also of non-cholinergic nerves. The possibility that such a second excitatory transmitter could be a catecholamine, 5-hydroxytryptamine, bradykinin or histamine has not been supported by experimentation (2). Burnstock, Dumsday and Smythe (3) have presented evidence that ATP might be the second transmitter in the bladder. In the present study we are investigating the role, if any, of prostaglandins in the non-cholinergic responses of urinary bladder to transmural stimulation. For this purpose, we have studied the effects of indomethacin, an inhibitor of prostaglandin synthesis, on responses to transmural stimulation.     

Percutaneous cryotherapy for metastatic bladder cancer: Experience with 23 patients

Bladder cancer is the most common malignancy of the urinary tract and in many patients is metastatic at diagnosis. Chemotherapy is the standard treatment for these patients but has serious side effects and in many patients is not tolerated. To avoid the side effects of systemic chemotherapy, patients with late stage bladder cancer have sought cryotherapy in our hospital. We reviewed data for the past 4 years to evaluate the safety and efficiency of percutaneous cryotherapy in 23 patients. Within 3 days after cryosurgery, all complications of bladder cancer (e.g. hematuria, urinary irritation, hypogastralgia, lumbago) had decreased to some degree. No new complications (e.g. bladder perforation) occurred and all complications had disappeared completely after 2 weeks. The progression-free survival (PFS) of these patients was 14 ± 8 months. There was no effect on PFS of tumor location or histopathology; however, differentiation status and tumor size influenced the therapeutic effect of percutaneous cryoablation. In conclusion, percutaneous cryotherapy may be a safe and efficacious therapeutic option in the treatment of metastatic bladder cancer.     

Genomic conservation of cattle microsatellite loci in wild gaur (Bos gaurus) and current genetic status of this species in Vietnam

Background

Cigarette smoking and chemical occupational exposure are the main known risk factors for bladder transitional cell carcinoma (TCC). Oxidative DNA damage induced by carcinogens present in these exposures requires accurate base excision repair (BER). The XRCC1 protein plays a crucial role in BER by acting as a scaffold for other BER enzymes. Variants in the XRCC1 gene might alter protein structure or function or create alternatively spliced proteins which may influence BER efficiency and hence affect individual susceptibility to bladder cancer. Recent epidemiological studies have shown inconsistent associations between these polymorphisms and bladder cancer. To clarify the situation, we conducted a comprehensive analysis of 14 XRCC1 polymorphisms in a case-control study involving more than 1100 subjects.

Results

We found no evidence of an association between any of the 14 XRCC1 polymorphisms and bladder cancer risk. However, we found carriage of the variant Arg280His allele to be marginally associated with increased bladder cancer risk compared to the wild-type genotype (adjusted odds ratio [95% confidence interval], 1.50 [0.98–2.28], p = 0.06). The association was stronger for current smokers such that individuals carrying the variant 280His allele had a two to three-fold increased risk of bladder cancer compared to those carrying the wildtype genotype (p = 0.09). However, the evidence for gene-environment interaction was not statistically significant (p = 0.45).

Conclusion

We provide no evidence of an association between polymorphisms in XRCC1 and bladder cancer risk, although our study had only limited power to detect the association for low frequency variants, such as Arg280His.     

A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer

Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.     

Implementation of a molecular epidemiology approach to human pleural malignant mesothelioma

The carcinogenic effect of asbestos has been reported in the literature since 40 years, and early studies describing the epidemic occurrence of malignant mesothelioma (MM) in asbestos workers, have become a paradigm of occupational cancer research. Research on MM was abandoned for many years since MM was considered as an asbestos-related disease, interesting only from a perspective of disease control and preventive policies. The introduction of new biological endpoints in the epidemiological studies has boosted research in the field, providing new tools for the study of emerging priority in cancer research and in public health. This approach, known as molecular epidemiology has a great potential in the study of MM, contributing to the understanding of susceptibility factors, to the evaluation of cancer risk in people occupationally or environmentally exposed to carcinogens, and to the enhancement of diagnosis and therapy. A comprehensive approach based on the use of banks of biological samples is presented and its advantages discussed here. The application of innovative endpoints, such as oncoproteins in biologic fluids, genetic polimorphisms, or gene function is discussed, and relevant literature reviewed.     

Comparative investigation of multiple organs of mice and rats in the comet assay

Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro-o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and D-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p,p'-DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p,p'-DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.     

Cell cycle kinetics of regenerating urothelial cells of the rat urinary bladder after partial cystectomy

The cell cycle kinetics of bladder urothelial cells regenerating after partial cystectomy were investigated in 96 female Wistar rats using the percentage labelled mitoses method. In the area of resection a mean cell cycle time (TC) of 15 h was determined. The DNA synthesis phase (TS) lasted 6 h and the premitotic-postsynthetic phase together with the mitosis phase (TG2 + M) 1.5 h, thus giving a presynthetic-postmiotic phase (TG1) of 7.5 h. Similar values were found for the urothelial cells in the stump: the mean cycle time measured 14 h, the TS-phase 6 h, the TG6 + M-phase 2 h and the TG1-phase 6 h. These data are discussed with respect to known cell cycle parameters of bladder urothelium regenerating in response to cytotoxic agents and of neoplastic urothelial cells. The reported findings provide a basis for further investigations using weak carcinogens and threshold doses of potent carcinogens to test the working hypothesis that stimulation of proliferation following partial cystectomy is capable of initiating, accelerating and/or potentiating carcinogenic cell transformation in the urinary bladder.     

Chemoprevention of tumors: the role of RAR-beta

Chemoprevention can be defined as the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. The knowledge of carcinogenic mechanisms provides the scientific rationale for chemoprevention. Epithelial carcinogenesis proceeds through multiple discernible stages of molecular and cellular alterations. Understanding of the multistep nature of carcinogenesis has evolved through highly controlled animal carcinogenesis studies, and these studies have identified three distinct phases: initiation, promotion and progression. Animal model studies have provided evidence that the development of cancer involves many different factors, including alterations in the structures and functions of different genes. Transitions between successive stages can be enhanced or inhibited in the laboratory by different types of agents, such activities providing the fundamental basis for chemoprevention.     

Crucial Roles of microRNA-Mediated Autophagy in Urologic Malignancies

Urologic oncologies are major public health problems worldwide. Both microRNA and autophagy, separately or concurrently, are involved in a variety of the cellular and molecular processes of multiple cancers, including urologic malignancies. In this review, we have summarized the related studies and found that microRNA-mediated autophagy acted as carcinogenic factors or suppressors in prostate cancer, kidney cancer, and bladder cancer. MiRNAs, targeted genes, and the different signaling pathways constitute a complex network that orchestrates autophagy regulation, militating the oncogenic and tumor-suppressive effects in urologic malignancies. Aberrant expression of miRNAs may induce the dysregulation of the autophagy process, resulting in tumorigenesis, progression, and resistance to anticancer therapies. Targeting specific miRNAs for autophagy modulation may present as reliable diagnostic and prognostic biomarkers or promising therapeutic strategies for urologic oncologies.     

Carcinogenesis: Mechanism and Criteria of Carcinogenic Activity

The pattern of carcinogenic action is described for locally acting as well as for remotely acting carcinogens. Whether applied locally, injected subcutaneously or given by mouth, aromatic polycyclic hydrocarbons are potentially carcinogenic for all tissues, whereas other known locally acting carcinogens have no such wide action. Solubility and diffusibility of the compound should be considered, but the question of localization of induced tumours is a problem of dose-response relationship.The method for evaluating carcinogenic action of a compound is based on the readiness with which the tumour is induced, and not in terms of the intensity of the response. The average latent period and the percentage of tumour yield are the two measures used. Since, at the present time, no chemical tests are available to determine whether a substance is carcinogenic, one must resort to biological methods of testing carcinogenic activity.The value of a long-term test for carcinogenesis, under a given set of rigidly controlled conditions, is emphasized, since there are so many variables which singly or in combination may alter the final effect of a given substance.