SYTTY
CHLORINATED IMPURITIES IN DRINKING WATER - MUTAGENIC COMPOUNDS AND THE MECHANISMS OF CANCER
Project leader: Hannu Komulainen, Laboratory of Toxicology, National
Public Health Institute, P.O.Box 95, FIN-70701 Kuopio, Finland, tel. +358-17-201
322, fax: +358-17-201 265, e-mail: Hannu.Komulainen@ktl.fi
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Researchers:
Pasi Hakulinen, Laboratory of Toxicology, National Public Health Institute,
Kuopio, Finland, tel. +358-17-201 324, Pasi.Hakulinen@ktl.fi
Veli-Matti Kosma, Department of Pathology and Forensic Medicine University
of Kuopio, Kuopio, Finland, tel. +358-17-162750, e-mail: Kosmave@jalus.uku.fi
Leif Kronberg, Department of Organic Chemistry, Åbo Akademi University,
Turku Finland, tel. +358-2-2154138, e-mail: Leif.Kronberg@abo.fi
Mariitta Laaksonen, Laboratory of Toxicology, National Public Health
Institute, Kuopio Finland, tel. +358-17-201 307, e-mail: Mariitta.Laaksonen@ktl.fi
Jorma Mäki-Paakkanen, Laboratory of Toxicology, National Public
Health Institute, Kuopio, Finland, tel. +358-17-201 345, e-mail:
Jorma.Maki-Paakkanen@ktl.fi
Kristina Servomaa, North Savo Regional Environment Centre, Kuopio,
Finland, tel. +358-17- 7884900, e-mail: Kristina.Servomaa@vyh.fi
Ritva Vasara, North Savo Regional Environment Centre, Kuopio, Finland,
tel. +358-17- 7884916, e-mail: Ritva.Vasara@vyh.fi
Raimo K. Tuominen, Department of Pharmacy, Division of Pharmacology
and Toxicology, University of Helsinki, Helsinki, tel. +358-9-70859469,
e-mail: RTuominen@helsinki.fi
Consortium: Drinking water and health
Financing SYTTY organisation: Tekes, The Academy of Finland
Funding from SYTTY / Total funding of project (€): 153000
/ 461682
Person-months of work funded by SYTTY / Total person-months of work:
49 / 99,5
KEY WORDS: Drinking water, chlorination, MX, tumor development,
p53.
EXTENDED ABSTRACT
1 Introduction
Chlorination is, and will be, the most common technique to disinfect drinking water worldwide. Unfortunately, chlorinated impurities are formed in water during the chlorination process from organic matter. Epidemiological evidence indicates that the long-term use of chlorinated drinking water is associated with cancer. The risk seems to be the lower the lower the mutagenicity of the water. The carcinogenic by-products in such water are not yet known. However, their identification would be important for the health risk assessment and for development of better water purification and disinfection processes.
MX (3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone) is the main mutagen in chlorinated water assessed by the Ames bacterial test (TA100 strain). It explains up to 60 % of the mutagenicity of chlorinated water and has to be considered as one candidate in the cancer risk. We have shown that MX is a multisite carcinogen in rats (Komulainen et al., J. Natl. Cancer Inst. 89:848-856, 1997). It caused tumors particularly in the liver and thyroid glands. The mechanisms of the tumorigenesis are not known but they were studied in the present study. They bear relevance because genotoxic carcinogens may be a health hazard already at low exposure levels upon continuous exposure. Evidence on genotoxic damage at the DNA level and cancer genes in MX-induced tumors and exclusion of the most imminent unspecific promotional mechanisms would provide credence that MX conrtibutes to the cancer risk in man.
Chlorinated drinking water also contains other chlorinated furanone-byproducts at the same or even higher concentrations as MX. Their genotoxicity in mammalian cells and potential to contribute to cancer risk is poorly characterized. Further characterization was executed in the present study.
2 Methods
In order to elucidate the mechanisms of the carcinogenesis of MX in rats, we analysed1 mutations in the genes known to be involved in carcinogenesis (p53, Ki-ras, Ha-ras and N-ras), and expression of the p53 protein in the liver tumors of animals of our previous carcinogenicity study. Mutations were screened utilizing TGGE- or SSCP-PCR techniques and direct sequencing. Expression of the p53 protein was analysed by immunohistochemistry, also in thyroid gland tumors2. Ki-ras expression was evaluated only in thyroid gland tumors.2
In other experiments3, hormonal effects of MX were studied by exposing rats with a single dose of MX, daily doses for one week or three weeks at three dose levels and measuring thyroid stimulating hormone (TSH), the thyroid gland hormones thyroxine (T4) and triiodothyronine (T3), prolactin and growth hormone in blood. An increase in TSH and a decrease in thyroid hormones would support the possibility that thyroid gland tumors were caused by hormonal promotion. Morphological changes in thyroid glands were evaluated by conventional histopathology and cell proliferation with immunohistological staining of PCNA, the proliferating cell nuclear antigen. The expression of p53 protein in the livers of these animals was also analysed1 to see whether MX affects its expression after short term exposures, in younger animals.
The genotoxicity of the structural congeners of MX, 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) was evaluated in Chinese hamster ovary cells (CHO) in vitro by analysing DNA damage using Comet assay, sister chromatid exchange (SCE) formation and chromosome aberrations.4
Since the mutation analyses and the immunohistochemistry did not show
evidence on genotoxicity of MX in tumor formation, the potential
of MX to promote malignant transformation was evaluated in two-stage
cell transformation assay in C3H10T1/2 cells.5
MX was evaluated both as an “initiator” and “promoter” in the assay.
3 Results and discussion
Molecular biological analyses of the MX-induced liver tumors in rats revealed only few point mutations.1 Altogether 4 point mutations were found in p53, all in tumors of the high dose group females of the MX study, but they had no correlation to tumor type or malignancy. The mutations were the type MX has caused in DNA in vitro but they were not the most typical ones. No point mutations were observed in ras genes in the liver tumors.
The p53 protein was overexpressed in all tumors of the bile duct epithelial cell origin in the liver but also in hyperplastic bile duct epithelial cells, independent on MX exposure.1 The as yet unknown factors maintained overexpression of the p53 protein in bile duct epithelial cells in aged rats. MX did not induce p53 expression after short term exposures in the liver.3
The proteins p53 and Ki-ras were not overexpressed in the MX-induced thyroid gland tumors.2 Mutation analysis on the respective genes was attempted but failed (too scarce tumor material, DNA no more solidly extractable in formalin-fixed samples).
The results on the mutation and protein expression analyses suggest that p53 and ras genes were not target genes of MX in the development of the MX-induced liver and thyroid gland tumors.
MX did not affect blood TSH and thyroid hormone levels in rats, in either sex, up to 3-week exposure.3 These data strongly suggest that the TSH-mediated hormonal promotion is not the key mechanism by which MX causes thyroid gland tumors in rats. MX did neither affect blood prolactin levels but slightly decreased growth hormone levels in males.
All chlorinated furanones induced DNA damage, SCEs and chromosome aberrations (mainly chromatid-type breaks and exchanges) in CHO cells in vitro.4 The order of potency, considering the lowest effective concentrations tested, to induce DNA damage (Comet assay) was CMCF.MCA.MX>MCF. The order to induce SCEs was MX>CMCF>MCA>MCF and to cause chromosome aberrations MX>MCA>CMCF>MCF. The levels of active concentrations did not differ much between MX, CMCF and MCA but was higher for MCF. The data show that also the chlorinated structural congeners of MX are genotoxic in mammalian cells in vitro, at about the same or higher concentration range than MX.
In the cell transformation assay in vitro, MX added alone at the initiation phase increased slightly the number of malignant transformation foci5 suggesting that it had genotoxic potential. When MX was added in the promotion phase of the assay, after treatment of the cells with 3-methylcholanthrene in the initiation phase, MX increased the number of malignant transformation foci. This may mean that MX acted as a tumor promoter in the assay but the exact cellular mechanisms need to be resolved for a firmer conclusion.
4 Conclusions
1. p53 and ras may not be target genes in MX-induced tumor development
in rats although they are mutated by several other carcinogens.
2. The development of the MX-induced thyroid gland follicular tumors
is not mediated by overproduction of the TSH hormone, the most typical
mechanism of thyroid gland carcinogenesis in rats.
3. MX may act as a tumor promoter but further knowledge on the cellular
mechanisms are needed for firmer conclusion.
4. In addition to MX, several other chlorinated furanone by-products
are genotoxic in mammalian cells in vitro, with a comparable potency. The
whole group should be considered together in assessment of the cancer
risk of chlorinated drinking water.