Autism and de novo Mutations

Advances in Autism Research
compiled by Teresa Binstock for ARI
April 2008

Autism and de novo Mutations

This past year, news articles have reported associations between de novo (non-inheritied) mutations and autism. These findings differ from those documenting associations between autism and inherited genetic syndromes such as Fragile X. What most such news articles didn't mention is that pollutants are a primary cause of de novo mutations. The first two citations report and discuss de novo mutations in relation to autism. The rest of the citations link de novo mutations with various pollutants.

De novo mutations and autism

1. Strong association of de novo copy number mutations with autism

Sebat J et al.
Science. 2007 Apr 20;316(5823):445-9.

We tested the hypothesis that de novo copy number variation (CNV) is associated with autism spectrum disorders (ASDs). We performed comparative genomic hybridization (CGH) on the genomic DNA of patients and unaffected subjects to detect copy number variants not present in their respective parents. Candidate genomic regions were validated by higher-resolution CGH, paternity testing, cytogenetics, fluorescence in situ hybridization, and microsatellite genotyping. Confirmed de novo CNVs were significantly associated with autism (P = 0.0005). Such CNVs were identified in 12 out of 118 (10%) of patients with sporadic autism, in 2 out of 77 (3%) of patients with an affected first-degree relative, and in 2 out of 196 (1%) of controls. Most de novo CNVs were smaller than microscopic resolution. Affected genomic regions were highly heterogeneous and included mutations of single genes. These findings establish de novo germline mutation as a more significant risk factor for ASD than previously recognized.
PMID: 17363630

2. Advancing paternal age and autism

Reichenberg A et al.
Arch Gen Psychiatry. 2006 Sep;63(9):1026-32.
http://archpsyc.ama-assn.org/cgi/content/full/63/9/1026

CONTEXT: Maternal and paternal ages are associated with neurodevelopmental disorders. OBJECTIVE: To examine the relationship between advancing paternal age at birth of offspring and their risk of autism spectrum disorder (ASD). DESIGN: Historical population-based cohort study. SETTING: Identification of ASD cases from the Israeli draft board medical registry. PARTICIPANTS: We conducted a study of Jewish persons born in Israel during 6 consecutive years. Virtually all men and about three quarters of women in this cohort underwent draft board assessment at age 17 years. Paternal age at birth was obtained for most of the cohort; maternal age was obtained for a smaller subset. We used the smaller subset (n = 132 271) with data on both paternal and maternal age for the primary analysis and the larger subset (n = 318 506) with data on paternal but not maternal age for sensitivity analyses. MAIN OUTCOME MEASURES: Information on persons coded as having International Classification of Diseases, 10th Revision ASD was obtained from the registry. The registry identified 110 cases of ASD (incidence, 8.3 cases per 10 000 persons), mainly autism, in the smaller subset with complete parental age data. RESULTS: There was a significant monotonic association between advancing paternal age and risk of ASD. Offspring of men 40 years or older were 5.75 times (95% confidence interval, 2.65-12.46; P<.001) more likely to have ASD compared with offspring of men younger than 30 years, after controlling for year of birth, socioeconomic status, and maternal age. Advancing maternal age showed no association with ASD after adjusting for paternal age. Sensitivity analyses indicated that these findings were not the result of bias due to missing data on maternal age. CONCLUSIONS: Advanced paternal age was associated with increased risk of ASD. Possible biological mechanisms include de novo mutations associated with advancing age or alterations in genetic imprinting.
PMID: 16953005

De novo mutations and pollutants

3. Will we be taught ethics by our clones? The mutations of the living, from endocrine disruptors to genetics

Vandelac L, Bacon MH.
Baillieres Best Pract Res Clin Obstet Gynaecol. 1999 Dec;13(4):571-92.

Considering the worldwide threat to health and reproduction related to endocrine disruptors (by-products of the chemical industry); considering the untrammelled development of the industrialization and engineering of the living, ethics and gynaecology/obstetrics itself is at a crossroads. Endocrine disruptors (derived from organochlorines and persistent organic pollutants such as PCBs, dioxins and furans, and pesticides such as aldrin, chlordane and DDT), are prime suspects in the deterioration of fertility and intellectual faculties and possibly a key factor in endometriosis, breast cancer and prostate cancer. The long-term and pernicious impacts of endocrine disruptors show our poor understanding of the complexities of life's mechanisms. Paradoxically, with our short-term perspectives and predilection for a technological fix, the problem posed by endocrine disruptors may accelerate the use of reproductive technologies such as ICSI and even cloning, as well as the dissemination of genetically modified organisms. The cure could be worse than the disease. Given the gravity of the challenge to humanity related to the chemical erosion of human health, the mutation of human conception introduced by reproductive technologies and by the drive to genetically modify nature and even human nature, we must urgently re-evaluate the direction in which our societies are headed and the reliance on profit-oriented technology to save us from ourselves. In these circumstances, the collective exercise of wisdom, prudence and responsibility towards the essence and integrity of humanity has become, more than ever, an ethical, and perhaps even a survival, imperative.
PMID: 10718712

4. Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes

Westphal GA et al.
Arch Toxicol. 2003 Jan;77(1):50-5.

Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 micro g/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 micro g/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.
PMID: 12491041

5. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location

Yauk C et al.
Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):605-10.
http://www.pnas.org/cgi/content/full/105/2/605

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.
PMID: 18195365

6. Possible involvement of oxidative stress in trichloroethylene-induced genotoxicity in human HepG2 cells
[Trichloroethylene is associated with autism (Windham et al 2006)]

Hu C et al.
Mutat Res. 2008 Mar 29;652(1):88-94.

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.
PMID: 18289923

7. Methylmercury genotoxicity: a novel effect in human cell lines of the central nervous system

Crespo-López ME et al.
Environ Int. 2007 Feb;33(2):141-6.

Methylmercury is an important source of environmental contamination and the central nervous system (CNS) is one of the main target organs. Methylmercury genotoxicity was already demonstrated in peripherical tissues but was never detected in the brain. Thus, the objective of this work was to verify its genotoxic effect using brain cell lines. Glioblastoma (U373) and neuroblastoma (B103) human cell lines were exposed to methylmercury (0-10 microM). By measuring cellular viability, concentrations inducing <20% of cellular death (P<0.05) were selected: 1 and 0.1 microM. To detect micronuclei, 200,000 cells were treated with methylmercury for 24 h, and then incubated with cytochalasin B (2 microg/ml) for 72 h (U373) or 48 h (B103). The binucleation index, frequency of micronucleated cells, micronucleation index, metaphasic index and index of nucleoplasmic bridges were determined. Statistical analysis showed indices and percentages significantly higher (P<0.05) in methylmercury-treated cells. Each cell line was shown to be differently sensitive to each biomarker of genotoxic damage, which seems to indicate the existence of different mechanisms of toxicity. This work demonstrates, for the first time, MeHg ability to provoke genotoxicity in cells of brain origin with relatively low levels of exposure.
PMID: 17007929

8. Genotoxicity of inorganic mercury salts based on disturbed microtubule function

Bonacker D et al.
Arch Toxicol. 2004 Oct;78(10):575-83.

[Burbacher et al 2005 found that thimerosal transitions to inorganic mercury in primate brains.]

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 microM, and inhibition is complete at about 10 microM. In this range, the tubulin assembly is fully (up to 6 microM) or partially (~6-10 microM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 microM Hg2+, the IC50 5.8 microM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 microM and a complete inhibition is reached at 1 microM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 microM HgCl2. Between 15 and 20 microM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 microM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 microM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.
PMID: 15205888

9. Genotoxicity of mercury used in chromosome aberration tests
[amalgam]

Akiyama M, Oshima H, Nakamura M.
Toxicol In Vitro. 2001 Aug-Oct;15(4-5):463-7.

The purpose of this study was to investigate the genotoxic effects of Hg released from dental amalgams. The chromosome aberration test was conducted using original extracts and their diluted solutions of conventional type amalgam and high copper amalgam. The concentrations of Hg, Cu and Ag in the original extract of high copper amalgam were 17.64, 7.97 and 43.90 microM, respectively. Those in the original extract of conventional type amalgam were 20.63, 7.87 and 14.79 microM, respectively. 10 and 30 microM Hg(2+) were also used for comparison. The frequency of chromosome aberrations was below 5% with 0 microM Hg(2+) and with a triple dilution of high copper amalgam extract, containing 5.88 microM Hg, 14.63 microM Cu and 2.65 microM Ag. However, 9.5% of the cells showed chromosome aberrations with a quadruple dilution of conventional type amalgam, containing 5.15 microM Hg, 3.69 microM Cu and 1.96 microM Ag. The amount of Hg in the quadruple dilution of conventional type amalgam was less than that in the triple dilution of high copper amalgam extract and 10 microM Hg(2+). A concentration of 30 microM Hg(2+) caused 34.5% of the cells to show chromosome aberrations while with a two-thirds dilution of high copper amalgam extract, containing 11.76 microM Hg, 29.26 microM Cu and 5.31 microM Ag, 58.5% of the cells showed chromosome aberrations. A two-thirds dilution of high copper amalgam extract induced more chromosome aberrations than 30 microM Hg(2+), although the amount of Hg was less than 30 microM Hg(2+). A triple dilution of conventional type amalgam extract, original extracts of conventional type amalgam and high copper amalgam and 100 microM Hg(2+) were induced few metaphases. It was revealed that the conventional type amalgam induced chromosome aberrations with quadruple dilution where cell viability was about 80% and that the high copper amalgam induced a high level of chromosome aberrations with the two-thirds dilution. The effects of low level Hg on humans are not clear.
PMID: 11566579

10. Protective effect of vitamin E on genotoxicity of methylmercury

Gilbert MM, Sprecher J, Chang LW, Meisner LF.
J Toxicol Environ Health. 1983 Oct-Dec;12(4-6):767-73.

The cytogenetic effects of methylmercury (MeHg) producing chromosomal breakages, C-mitosis, and sister chromatid exchange are well documented. The present investigation was to determine whether vitamin E, which has been shown to exert a certain protective measure on the nervous system of animals exposed to methylmercury, also will diminish the genotoxicity of MeHg. Adult hamsters were given a daily injection (ip) of methylmercury chloride at a dosage of 2.0 mg/kg body weight (kg b.w.) and/or with 2.0 mg/kg b.w. vitamin E for 3 wk. All animals were sacrificed 5 d after the last day of MeHg exposure. Fibroblast cultures were established with cells obtained from skins of these animals. Chromosome preparations were made from these cultures and evaluated, using a double-blind system, for incidences of chromosomal breakage. It was found that all the fibroblast cultures grew well except from those animals treated with MeHg. Furthermore, while the saline control and vitamin E alone cultures showed no significant number of chromosomal breakages, cells from animals treated with only MeHg showed a high incidence of chromosomal damage (50% with 1 break and 15% with 2 or more breaks). Such chromosomal damage, however, was totally eliminated by vitamin E treatment (no chromosomal breakage was observed in cultures from MeHg/vitamin E animals). Our present study represents the first report on in vitro chromosomal damages as a result of in vivo MeHg exposure. Furthermore, our investigation also demonstrated the protective potential of vitamin E against genotoxicity of methylmercury.
PMID: 6668622

11. The influence of environmental exposure to complex mixtures including PAHs and lead on genotoxic effects in children living in Upper Silesia, Poland

Mielzyńska D, Siwińska E, Kapka L, Szyfter K, Knudsen LE, Merlo DF.
Mutagenesis. 2006 Sep;21(5):295-304.

Environmental exposure is a complex mixture of hazardous compounds with different mechanisms of toxicity. In case of concomitant exposure to carcinogenic substances--such as polycyclic aromatic hydrocarbons (PAHs)--and to heavy metals--such as lead (Pb)--the level of DNA damage may be enhanced. Children are considered more vulnerable than adults to chemical toxicants because they take in more toxicants as a proportion of body mass and because of inherent biological growth and developmental factors. The objective of the study was to measure cytogenetic effects in Silesian children and to investigate their relation with the environmental exposure to PAHs and Pb. The examined population included 74 children 5-14-year-old who lived in two cities located in the most polluted centre of the Silesia province. Individual exposure to lead was assessed for each child by measuring lead in blood (PbB), and to PAH by measuring 1-hydroxypyrene in urine (1-OHP), urinary mutagenicity and DNA adducts in circulating lymphocytes. Biomarkers of genetic effects were assessed by measuring micronuclei (MN) and sister chromatid exchanges (SCE) in children's peripheral lymphocytes. The mean levels of biomarkers of exposure were as follows: PbB 7.69 microg/dl, DNA adducts 9.59 adducts per 10(8) nt, 1-OHP 0.54 micromol/mol creatinine, and urinary mutagenicity presented as the number of revertants per mmol of creatinine: 485 for TA 98 and 1318 for YG1024. Mean value of MN was 4.44 per 1000 binucleated cells and SCE frequency ranged between 6.24 and 10.06 with a mean value of 7.87. The results suggest the influence of exposure to environmental agents on the induction of cytogenetic effects in peripheral lymphocytes of children: namely Pb on MN and PAHs on SCE. The sources of that exposure may be outdoor and indoor. Emissions from coal-burning stoves are important contributors to the total exposure to PAHs and Pb in Silesian children.
PMID: 16891332

12. Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide

Lambert B, Andersson B, Bastlova T, Hou SM, Hellgren D, Kolman A.
Environ Health Perspect. 1994 Oct;102 Suppl 4:135-8.
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1566944&blobtype=pdf

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.
PMID: 7821287

13. Is there a causal association between genotoxicity and the imposex effect?

Hagger JA et al.
Environ Health Perspect. 2006 Apr;114 Suppl 1:20-6.
http://www.ehponline.org/members/2005/8048/8048.html

There is a growing body of evidence that indicates common environmental pollutants are capable of disrupting reproductive and developmental processes by interfering with the actions of endogenous hormones. Many reports of endocrine disruption describe changes in the normal development of organs and tissues that are consistent with genetic damage, and recent studies confirm that many chemicals classified to have hormone-modulating effects also possess carcinogenic and mutagenic potential. ...histological examination of a larger number of dog-whelks collected from sites throughout Europe confirmed the presence of hyperplastic growths, primarily on the vas deferens and penis in both TBT-exposed male snails and in females that exhibited imposex. A strong association was found between TBT body burden and the prevalence of abnormal growths, thereby providing compelling evidence to support the hypothesis that environmental chemicals that affect reproductive processes do so partly through DNA damage pathways.
PMID: 16818242

14. Molecular mechanism of genotoxicity of the environmental pollutant 3-nitrobenzanthrone
["identified in diesel exhaust and air pollution"]

Stiborová M et al.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005 Dec;149(2):191-7.
http://publib.upol.cz/%7Eobd/fulltext/Biomed/2005/2/191.pdf

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen identified in diesel exhaust and air pollution. This article reviews the results of our laboratories showing which of the phase I and II enzymes are responsible for 3-NBA genotoxicity, participating in activation of 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), to species generating DNA adducts. Among the phase I enzymes, the most of the activation of 3-NBA in vitro is attributable to cytosolic NAD(P)H:quinone oxidoreductase (NQO1), while N,O-acetyltransferase (NAT), NAT2, followed by NAT1, sulfotransferase (SULT), SULT1A1 and, to a lesser extent, SULT1A2 are the major phase II enzymes activating 3- NBA. To evaluate the importance of hepatic cytosolic enzymes in relation to microsomal NADPH:cytochrome P450 (CYP) oxidoreductase (POR) in the activation of 3-NBA in vivo, we treated hepatic POR-null and wild-type C57BL/6 mice with 3-NBA or 3-ABA. The results indicate that 3-NBA is predominantly activated by cytosolic nitroreductases such as NQO1 rather than microsomal POR. In the case of 3-ABA, CYP1A1/2 enzymes are essential for the oxidative activation of 3-ABA in liver. However, cells in the extrahepatic organs have the metabolic capacity to activate 3-ABA to form DNA adducts, independently from CYP-mediated oxidation in the liver. Peroxidases such as prostaglandin H synthase, lactoperoxidase, myeloperoxidase, abundant in several extrahepatic tissues, generate DNA adducts, which are formed in vivo by 3-ABA or 3-NBA. The results suggest that both CYPs and peroxidases may play an important role in metabolism of 3-ABA to reactive species forming DNA adducts, participating in genotoxicity of this compound and its parental counterpart, 3-NBA.
PMID: 16601755

15. Genotoxic effects of rotenone on cultured lymphocytes

de Lima PD et al.
Genet Mol Res. 2005 Dec 30;4(4):822-31.

Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle.
PMID: 16475130

16. Hospital wastewater genotoxicity

Jolibois B, Guerbet M.
Ann Occup Hyg. 2006 Mar;50(2):189-96.
http://annhyg.oxfordjournals.org/cgi/content/full/50/2/189

Hospitals represent an incontestable release source of many chemicals compounds in their wastewaters, and which may have an impact on the environment and human health. Indeed, some of the substances found in wastewaters are genotoxic and are suspected to be a possible cause of the cancers observed in the last decades. To study the toxicity and the risk associated with these releases biological tests, such as genotoxicity tests, can be used. An evaluation of the genotoxic potential of the wastewaters from a university hospital was performed with the SOS chromotest and the Salmonella fluctuation test. The samples were taken for six 1-week periods between May 2001 and April 2003. Out of a total of 38 samples tested, 31 were positive in at least one assay (82%). Distribution, proportion and intensity of the genotoxic response were different among the six sampling periods. The two genotoxicity tests had different sensitivities. It must be emphasized that whatever the sampling period, Monday samples were always genotoxic in at least one assay. This work shows that this hospital wastewaters samples are very often genotoxic, the response intensity being inflected by rain levels. Efforts must be undertaken by hospitals to integrate the knowledge and the control of their wastewaters in infection and environmental control programs.
PMID: 16143715

17. Cytogenetic and genotoxic effects of the insecticides, imidacloprid and methamidophos

Karabay NU, Oguz MG.
Genet Mol Res. 2005 Dec 30;4(4):653-62.

We examined the cytogenetic and genotoxic effects of the neonicotinoid insecticide imidacloprid and the organophosphate insecticide methamidophos, when administered alone or in combination. These insecticides were tested with the bone marrow chromosome aberration assay and micronucleus test in rats and by the bacterial mutation assay (Salmonella/microsome mutagenicity assay). Wistar albino rats were orally fed daily with laboratory chow treated with various concentrations of insecticides, 50 and 100 mg/kg imidacloprid, 2.5 and 5 mg/kg methamidophos, and 2.5 and 5 mg/kg imidacloprid plus methamidophos, respectively, for 90 days. Numerical and structural chromosomal aberrations were evaluated. Significant differences were detected between all the insecticide-administered groups versus the control group and between the two concentrations of the pesticide-treated groups. Both concentrations of the insecticides induced a dose-related increase in the micronucleus frequency (P < 0.05). Dose-related increases in the number of revertants were observed with the two Salmonella strains (TA98 and TA100). All tested doses of the insecticides demonstrated mutagenic activity in the presence of S9 mix. These results lead us to the conclusion that the synergistic effect of methamidophos and imidacloprid causes an increase in potential damage to non-target organisms.
PMID: 16475109

18. Hormesis and dose-response-mediated mechanisms in carcinogenesis: evidence for a threshold in carcinogenicity of non-genotoxic carcinogens

Fukushima S et al.
Carcinogenesis. 2005 Nov;26(11):1835-45.
http://carcin.oxfordjournals.org/cgi/content/full/26/11/1835

Recently the idea of hormesis, a biphasic dose-response relationship in which a chemical exerts opposite effects dependent on the dose, has attracted interest in the field of carcinogenesis. With non-genotoxic agents there is considerable experimental evidence in support of hormesis and the present review highlights current knowledge of dose-response effects. In particular, several in vivo studies have provided support for the idea that non-genotoxic carcinogens may inhibit hepatocarcinogenesis at low doses. Here, we survey the examples and discuss possible mechanisms of hormesis using phenobarbital, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), alpha-benzene hexachloride (alpha-BHC) and other non-genotoxins. Furthermore, the effects of low and high doses of non-genotoxic and genotoxic compounds on carcinogenesis are compared, with especial attention to differences in mechanisms of action in animals and possible application of the dose-response concept to cancer risk assessment in humans. Epigenetic processes differentially can be affected by agents that impinge on oxidative stress, DNA repair, cell proliferation, apoptosis, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors, cause aberrant DNA methylation at the genomic level and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. Genotoxic agents, in contrast, cause genetic change by directly attacking DNA and inducing mutations, in addition to temporarily modulating the gene activity. Carcinogens can elicit a variety of changes via multiple genetic and epigenetic lesions, contributing to cellular carcinogenesis.
PMID: 15975961

19. Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Silva-Pereira LC et al.
Braz J Med Biol Res. 2005 Jun;38(6):901-7. Epub 2005 Jun 1.
http://www.scielo.br/

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 microg/l) of HgCl2 and CH3HgCl and incubated at 37 degrees C for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 microg/l alone, and at 1, 10, 100, and 1000 microg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.
PMID: 15933784

20. Genotoxicity and cytotoxicity assessment in lake drinking water produced in a treatment plant

Buschini A et al.
Mutagenesis. 2004 Sep;19(5):341-7.

Chemical analyses and short-term mutagenicity bioassays have revealed the presence of genotoxic disinfection by-products in drinking water. In this study, the influence of the different steps of surface water treatment on drinking water mutagen content was evaluated. Four different samples were collected at a full-scale treatment plant: raw lake water (A), water after pre-disinfection with chlorine dioxide and coagulation (B), water after pre-disinfection, coagulation and granular activated carbon filtration (C) and tap water after post-disinfection with chlorine dioxide just before its distribution (D). Water samples, concentrated by solid phase adsorption on silica C18 columns, were tested in human leukocytes and HepG2 hepatoma cells using the comet assay and in HepG2 cells in the micronuclei test. A significant increase in DNA migration was observed in both cell types after 1 h treatment with filtered and tap water, and, to a lesser extent, chlorine dioxide pre-disinfected water. Similar findings were observed for the induction of "ghost" cells. Overloading of the carbon filter, with a consequent peak release, might explain the high genotoxicity found in water samples C and D. Cell toxicity and DNA damage increases were also detected in metabolically competent HepG2 cells treated with a lower concentration of tap water extract for a longer exposure time (24 h). None of the water extracts significantly increased micronuclei frequencies. Our monitoring approach appears to be able to detect contamination related to the different treatment stages before drinking water consumption and the results suggest the importance of improving the technologies for drinking water treatment to prevent human exposure to potential genotoxic compounds.
PMID: 15388805

21. Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters

Georgiadis P et al.
Carcinogenesis. 2001 Sep;22(9):1447-57.
http://carcin.oxfordjournals.org/cgi/content/full/22/9/1447

The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydrocarbons (PAH) were significantly higher in Athens subjects during both seasons. There was hardly any diagonal radioactive zone in the pattern of DNA adducts observed. Highest adduct levels were observed in a sub-group of subjects living in or near the Halkida Institute campus, which was located in rural surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution.
PMID: 11532867

22. Molecular bases of hprt mutations in malathion-treated human T-lymphocytes

Pluth JM et al.
Mutat Res. 1998 Feb 2;397(2):137-48.

Recently, we reported that 6 of 84 (7.1%) hprt mutants arising in in vitro malathion-treated human T-lymphocytes were characterized by specific genomic deletions in a 125-bp region of exon 3 (Pluth et al., Cancer Research 56 (1996) 2393-2399. We have now extended study to determine whether additional differences in molecular spectrum at a basepair level exist between control and malathion-treated mutations, and investigated whether there is evidence to support the hypothesis that malathion is an alkylating agent. We analyzed 101 hprt mutants (24 from control and 77 from treated cultures) isolated form six in vitro malathion exposures of T-lymphocytes from four healthy male donors. Analysis consisted of: Southern blotting, genomic multiplex PCR, genomic DNA sequencing and reverse transcription of PCR amplification (RT/PCR) and sequencing of the cDNA product. Mutations at several basepair sites were frequent after malathion exposure and were isolated from treated cells from at least two different individuals. Using a human hprt mutation database for comparison, the frequency of mutations at one of these sites (basepair 134) was found to be significantly elevated in the malathion-treated cell (p < 0.0005). Hprt mutations in malathion-treated cells arose preferentially at G:C basepairs, which is consistent with earlier reports that malathion alkylates guanine nucleotides. Assessing molecular changes at both genomic and cDNA levels in the same mutants revealed that many small, partial exon deletions (< 20 bp) in genomic DNA were often represented in the cDNA at the loss of one or more exons. In addition, It was noted that identical genomic mutations can result in different cDNA products in different T-cell isolates. These observations affirm the importance of genomic sequence analysis in combination with RT/PCR for a more accurate definition of the mutation spectrum.
PMID: 9541638

23. Distinguishing potential sources of genotoxic exposure via HPRT mutations

Molholt B, Finette BA.
Radiats Biol Radioecol. 2000 Sep-Oct;40(5):529-34.

We utilize T-cell HPRT mutations to monitor exposure to environmental mutagens in siblings of children who have developed cancer at a persistently high rate in Toms River, New Jersey, U.S.A. A preliminary epidemiological study has found a statistically-significant association between drinking public water (by pregnant mother or infant) and subsequent risk for childhood cancer. Three potential sources of mutagenic exposures in Toms River may have increased the rate of carcinogenic initiation significantly in children: 1. Benzidine-based, other azo dye and anthraquinone dye wastes released by Ciba-Geigy, 2. Styrene-acrylonitrile (SAN) trimer and other plastic wastes of Union Carbide, and 3. Radium-224, present in unusually high concentrations in the Cohansey aquifer. Specific patterns of HPRT mutations are utilized to distinguish these various potential sources of carcinogenic exposures in the drinking water of families with childhood cancer and to differentiate chemically or radiologically induced cancers from those which occur spontaneously.
PMID: 11130945