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CARCINOGENICITY AND MUTAGENICITY OF MALATHION AND ITS TWO ANALOGUES: A SYSTEMATIC REVIEW
RESUMO

O agrotóxico malathion vem sendo amplamente utilizado no mundo em programas de controle de arboviroses e em 2015 foi classificado pela Agência Internacional para Pesquisas em Câncer (IARC) como provável agente carcinogênico para seres humanos. Este trabalho objetivou a sistematização das evidências dos efeitos carcinogênicos e mutagênicos associados à exposição do malathion e seus análogos, malaoxon e isomalathion. A busca foi realizada nas bases de dados TOXLINE, PUBMED e SCOPUS por artigos originais publicados de 1983 a 2015. Do total de 273 artigos elegíveis, foram selecionados 73. Os resultados dos estudos in vitro e in vivo evidenciaram danos genéticos e cromossômicos provocados pelo malathion; os estudos epidemiológicos evidenciaram associações significativamente positivas para cânceres de tireóide, de mama, e ovariano em mulheres na menopausa. Estas evidências do efeito carcinogênico do malathion devem ser considerados diante de sua utilização em programas de controle de arboviroses.
Palavras-chaves: Malation. Testes de Carcinogenicidade. Testes de Mutagenicidade. Saúde Ambiental.


ABSTRACT
Malathion has been widely used worldwide in arbovirus control programs. In 2015, it was classified by the International Agency for Research on Cancer (IARC) as a probable carcinogen to humans. This work aimed to systematize the evidence of the carcinogenic and mutagenic effects associated with the exposure of malathion and its analogs, malaoxon and isomalathion. The search was carried out in Toxline, PubMed and Scopus databases for original papers published from 1983 to 2015. In all, 73 papers were selected from a total of 273 eligible papers. The results of in vitro and in vivo studies showed mainly genetic and chromosomal damages caused by malathion. The epidemiological studies evidenced significant positive associations for thyroid, breast, and ovarian cancers in menopausal women. This evidence of the carcinogenic effect of malathion should be considered before its use in arbovirus control programs.
Keywords: Malathion. Carcinogenicity Tests. Mutagenicity Tests. Environmental Health.
INTRODUCTION
Pesticides are chemical compounds used extensively in agriculture, for the chemical control of spontaneous species in urban environment and vectors, in public health campaigns1 and are a danger to humans and nature2. Among the widely used is the group of organophosphates (OP), which are irreversible inhibitors of acetylcholinesterase (AChE), active in all animal groups that use acetylcholine as a neurotransmitter3. The consequent accumulation of this molecule in the body causes toxic effects on the different systems of exposed living beings, such as muscular, nervous, immune and endocrine alterations4-7. Among its possible chronic effects, characterized by late onset, are irreversible damages, such as paralysis and neoplasms8.
In Brazil, with the exponential growth of the dengue epidemic in 2015, when more than 1 million cases were confirmed9, and with the onset of Chikungunya fever (in 2014) and the Zika virus epidemic (in 2015), with consequences even more harmful to the population, a review of the National Aedes aegypti (transmitter mosquito) Control Program was requested, with the intensified use of larvicides and adulticides for this mosquito, going back to the 2014 guidelines regarding the use of UBV spraying with Malathion at 30% diluted in water for the whole national territory10. This was criticized by the Brazilian Association of Public Health, which issued a technical note warning of the dangers to health and the environment11.
Malathion (diethyl (dimethoxythiophosphorylthio) succinate) is an OP used in various food crops for the control of unwanted species and is often used in insect control12, as a commercial or technical quality product containing approximately 90-95% of the product in weight and may contain up to twelve impurities formed during manufacture and storage13. Among the relevant impurities are malaoxon and isomalathion, produced by oxidation14 and the chemical or thermal isomerization of malathion, respectively15.
Its mutagenic capacity and potential carcinogenic effect have been discussed16-18. However, despite widespread use, there are surprisingly few studies on the association of malathion and cancer, most of them in North America, others in Europe, while very few have been conducted in less industrialized countries where exposure is likely to be much higher19. Some authors point out their findings as something of concern since malathion has shown high levels of carcinogenic activity, as well as chemical properties that bring it closer to other notably carcinogenic substances such as aflatoxin and benzopyrene20.
In the 1980s, malathion was considered and evaluated by the International Agency for Research on Cancer (IARC) Working Group as not classifiable as to its carcinogenicity to humans (Group 3)21,22 because it concluded that there was insufficient evidence for the carcinogenicity of malathion or its metabolite malaoxon in experimental animals, and data for humans were not available at the time. However, in 2015, IARC published a new document, classifying the pesticide as a probable human carcinogen (Group 2A)19.
In view of the widespread use of malathion as a pesticide worldwide, both in agriculture and public health, and in view of the risks that it can bring, this work aimed to systematize the evidence of the carcinogenic and mutagenic effects associated with the exposure of this organophosphate pesticide and its analogues (malaoxon and isomalathion).
METHODS
Search data
A systematic review of the literature was performed by searching scientific papers published between 1983 and 2015. The year 1983 was chosen as the starting point of the research, since the IARC21 monograph published in that year considered malathion as “not classifiable as to its carcinogenicity to humans (Group 3)”, concluding that there was insufficient evidence for the carcinogenicity of malathion or its metabolite malaoxon in animal experiments, and there were no data regarding humans.
The search was performed in the electronic databases Scopus, PubMed and Toxline (in the latter the results of PubMed were excluded), and two command groups were employed. The first, consisting of terms related to the exposure of interest (malathion, malaoxon and isomalathion), and the second containing terms related to the outcome of interest (cancer, carcinogenicity tests, carcinogens, neoplasms, mutagenesis, mutagenicity tests and mutagens). A query was made in the Medical Subject Headings (MeSH) to select the descriptors/terms used. Boolean operator “OR” was used for the combination of the terms in each group, and “AND” operator was used for the combination between the groups.
Selection of papers
We selected original studies that showed results on the carcinogenic or mutagenic effect of malathion, malaoxon and isomalathion in living beings. Review papers, dissertations and theses identified in the search were excluded. English, Portuguese and Spanish manuscripts were considered.
The papers were selected by the researchers (authors of this paper) in two stages. In the first one, two researchers read separately the title and summary of the papers for the selection of those that should be the basis of the research. Those without an abstract or insufficient information to make a decision were kept into the next step. Cases of disagreement were resolved by a third researcher. In the second step, all papers selected that met the inclusion/exclusion criteria and those with insufficient information for decision-making were analyzed in their entirety. As in the previous stage, the data of both reviewers were again confronted, and the differences were solved by the third reviewer.
The selected manuscripts were analyzed for the extraction of the following data: authors, year of publication, journal, study design, target population, country of study population (in epidemiological studies), exposure (malathion, malaoxon, isomalathion, mixed exposure) and main results identified regarding the carcinogenic and mutagenic effect of malathion, malaoxon and isomalathion.
Search was performed between July 4 and 12, 2016. Filters were used to select the languages and the period of publication of the manuscripts in the three databases.
RESULTS AND DISCUSSION
Search returned 178 results in Scopus, 147 in PubMed and 92 in Toxline, totaling 417 papers (Figure 1). A total of 142 duplications were identified, as well as two papers published in other languages (German and Chinese), leaving out 273 papers for eligibility evaluation.
(FIGURE 1)
After applying the inclusion and exclusion criteria and the two-step evaluation, 73 papers were included in this study, all in English. Of these, 24 correspond to in vitro experimental studies, including mutagenicity tests and cell cultures of both animals and human beings; 24 in vivo experimental studies, including mice, rats, hamsters, birds, frogs and flies; 25 epidemiological studies, including cohorts, case-controls and cross-sectional studies (two of them with in vitro experiments as well). The results found in the in vitro experimental studies that evaluated the carcinogenic or mutagenic effect of malathion and its analogs are summarized in Table 1.
(CHART 1)
Three studies have shown significant increases in the DNA damage of rat peripheral blood lymphocytes following exposure to malathion1,18,26. Higher frequencies of chromosomal changes in bone marrow cells of mice in a dose-dependent manner to malathion were also observed26. Besides observing the significant reduction of cell viability and the significant increase of breaks in both single and double strand DNA, Ojha and Gupta18 verified the formation of DPC, in time- and dose-dependent fashion after exposure of rats’ lymphocytes to malathion both individually and mixed with two other organophosphates (Chlorpyrifos and Parathion-Methyl) when compared to the control. Since DPCs correspond to toxic lesions associated with the toxicity mechanism (s) of carcinogenic compounds45, the study shows the carcinogenic effect of malathion. The research further concludes that malathion, along with the other pesticides of the study, should generate oxidative lesions of DNA base pairs, with a genotoxic potential to alter enzyme expression.
In cultures of human peripheral blood lymphocytes exposed to malathion, results showed dose-dependent increase of chromosomal changes and sister chromatid exchanges37,39, as well as the alkylating effect of DNA-specific nucleotides34. No significant changes were observed in the DNA damage of these same cells32 when malathion was evaluated in comparison to its two analogs, unlike malaoxon and isomalathion, which induced DNA damage in a dose-dependent fashion. The study by Blasiak et al.32 was the only one to evaluate the three chemical agents in the same study and concluded that the damages caused to the DNA by malaoxon are more pronounced than those caused by isomalathion.
Josse et al.23 evaluated the effects of malathion and isomalathion individually and combined on the human liver HepaRG cell line and showed that, although isomalathion was much more cytotoxic than malathion, both substances showed similar mutagenic effects in these hepatocytes. On the other hand, Błasiak and Trzeciak33 claim that DNA damage to human lymphocytes is caused by isomalathion and not by its original compound.
The results are somewhat contradictory regarding malaoxon exposure. Blasiak and Kowalik31 found that malaoxon promoted a significant increase in the level of DNA damage in human peripheral blood lymphocytes in the Comet Assay, and was higher with increasing dose, while in the human lineage choriocarcinoma cell (JAR) assay (model acceptable for human placental cells), malathion, and not the same agent, was found to be responsible for the cytotoxic and genotoxic effects in these cells in vitro. Authors argue that metabolites of pesticides with contradictory results regarding their carcinogenic potential should be studied, since they may represent the true carcinogens46.
Malathion has also been evaluated in gene mutation tests that employ bacteria, such as the Salmonella lactam test (genotoxin detection method)35, phage-MicroScreen (a miniaturized system that uses the induction of prophylaxis in Escherichia coli X as an indicator of genetic damage)41 and the Ames Salmonella test40. In the three studies found, the results were negative for the mutagenic activity of malathion, and no mutagenic activities of malathion were observed in the last test before or after activation of the rat liver S9 fraction in the respective non-toxic doses (33 mg/L) and 90% toxic (1650mg/L). In a literature review, Flessel et al.47 also observed the same results and concluded that malathion does not appear to induce timely mutations in DNA in bacterial systems.
Of the 24 in vivo experimental studies found in this systematic review, twenty performed the experiments on rats, mice or hamsters, while the others evaluated the effects of the pesticide on birds, frog and flies. Table 2 shows the main results of these studies.
(CHART 2)

In 1984, Degraeve et al.70 reported that Dynafos and Phosan Plus, insecticides containing malathion in their composition, did not induce chromosomal changes in bone marrow cells, spermatogonia or primary spermatocytes, nor alterations in the dominant lethal mutation assay, after intraperitoneal injection of rats of strain Q. However, the study has no data regarding impurities, solvents, emulsions and the like in these compounds, which shows its limitation.
In the same year, these authors exposed rats from the same strain to malathion at 99% purity by ingestion of water containing small amounts of the organophosphate (8 ppm, corresponding to the highest value allowed in Belgium for pesticide residues in fruits and vegetables) five days a week for seven consecutive weeks69. Again, they did not observe a significant increase in chromosomal breaks or gaps in the same cell types from the previous work, nor in the dominant lethal mutation assay. However, there is hardly any information on the analytical technique used to search for these changes. In 1985, the group evaluated the effects on rats injected with malathion (150 mg/kg) combined with trichlorfon (50 mg/kg), and also did not show cytogenetic effects (breaks, gaps, and chromatid alterations) of the combination68.
However, of the total in vivo experimental studies found in this study, only four showed no positive association between mutagenic effects and malathion, all from the 1980s. The first three were those mentioned above, the fourth and last of them dating from 1987, which obtained negative results regarding the production of sexual chromosome losses and non-disjunction in strains of Drosophila melanogaster exposed to malathion with 50% of the active ingredient, a percentage that may have had a significant influence on the results found66. Since then, there have been a further 17 studies with positive results for carcinogenicity and genotoxicity of malathion, and one for malaoxon genotoxicity.
Honda and Sinha65 showed a significant increase (at a level of 0.1%) in the chromatid alterations of bone marrow cells after exposure of Mus musculus rats. Swiss albino mice strains exposed to malathion evidenced bone marrow cell metaphases with various types of chromosomal alterations, and the pesticide-induced significantly higher frequency of alterations (p<0.001) in the three acute doses tested (2.5, 5 and 10mg/kg) than in control, besides increased frequencies of sister chromatid exchanges and sperm head anomalies59. Giri et al.54 reported the dose-dependent increased frequency of micronucleated polychromatic erythrocytes in bone marrow cells of mice of the same species after in vivo exposure to malathion at 95% purity.
Regarding the research on the carcinogenicity of malathion in in vivo experiments, the first manuscript found dated back to 1992, in which the pesticide promoted a significant increase (p<0.05) in the induction of GST-P positive focus in hepatocytes of F344 mice compared to control64. The expression of the GST-P enzyme is usually low in fetal hepatocytes, quiescent adults or in regeneration, placenta, heart and other organs of male rats, however, hyperplastic nodules and chemically-induced liver tumors exhibit GST-P values of about 20 to 50 times and 10 to 30 times over, respectively, when compared to normal rat liver values72. Thus, the study suggests the possibility of malathion being a weak hepatocarcinogenic or hepatopromoting agent. On the other hand, the study by Hoshiya et al.63, also with F344 mice, concludes and states that malathion has liver tumor promoting activity.
Studies have investigated the carcinogenic effects of malathion and estrogen in virgin female rats of the Sprague-Dawley strain, individually and in combination. Calaf and Garrido55 observed progressive changes in the mammary cell ducts of rats treated with isolated malathion compared to control after 240 days of treatment. Besides the significantly increased size (p<0.05) of ducts in the mammary gland proliferation phase of rats treated with the pesticide, Calaf and Chau51 found a growing expression of the mutant p53 tumor marker protein. Several types of pre-neoplasms in the bronchiolar epithelium of rats injected with malathion have also been found, besides carcinomas in situ57. In the renal tissue of malathion-exposed rats, results suggest abnormalities with signs of malignancy52.
In most of the previously cited studies51,52,57, treatment with the combination of malathion and estrogen-induced more cellular alterations than treatments with the substances alone. Thus, the combination of pesticides found in the environment, widely used in Latin America and many other countries, and an endogenous substance, such as estrogen, has the potential to induce deleterious effects on humans, such as breast cancer, for example51. Estrogen can also be found as a pollutant in surface and groundwater, and its presence in the environment can have severe toxicological and ecotoxicological repercussions since this substance is recognized as an endocrine disrupter, associated with premature puberty, infertility and congenital malformation73-75.
The epidemiological studies found in this systematic review are summarized in Table 3 below. The types of designs obtained were eleven control case studies, eight cohorts and six cross-sectional studies, where two of these also performed in vitro experiments with human peripheral blood lymphocytes.
(CHART 3)
Most studies (n=16) investigated the correlations between exposure to malathion and the development of cancers. Of the 25 papers, 18 were conducted in North America (12 in the U.S. and 6 in Canada), three in Europe, three in Asia and only one in Latin America (Chile). These findings corroborate the IARC manuscript, which states that very few studies on this approach have been conducted in less industrialized countries19.
As to the evidence of genotoxicity investigated, in a cross-sectional study with male pesticide applicators, Andreotti et al.76 found positive associations between the recent use of malathion and the shorter telomere relative length (p=0.03). Telomere shortening is associated with several diseases, and most studies have reported associations between telomere length and increased risk of cancer101,102.
In a prospective cohort with patients who attempted suicide by self-poisoning with drugs or insecticides, temporary but significant increases in peripheral blood leukocyte aneuploidy (6.3%, p<0.01) and chromatid (5.3%, p<0.01) and chromosomic (1.4%, p<0.01) alteration rates after intoxication with malathion97 were observed. Also, one of 14 people poisoned by organophosphate died.
Only one of the epidemiological manuscripts addressing the mutagenic effects of malathion and complex mix of pesticides including the organophosphate did not find a positive association95. This work was carried out both with a cross-sectional epidemiological study of workers exposed to pesticides and with in vitro experimentation of human peripheral blood lymphocytes also concluded that malathion has a relatively low potential to cause chromosomal damage in vitro, and the corresponding doses used in the experiment were much higher than those that professional applicators are likely to be exposed to in vivo.
On the other hand, a cross-sectional study with a worker occupationally exposed to pesticides (primarily malathion and phosphine) for more than five years, and an experimental in vitro study with human peripheral blood lymphocytes exposed to malathion revealed that the mutagenicity of malathion can be detected at a molecular level96. Molecular changes in the hprt assay were observed at doses of malathion that did not produce in vitro cytotoxicity (≤50 mg/ml) and at exposure levels experienced by the individual from which the in vivo mutant was retrieved. The authors state that alterations similar to those reflected in the hprt in this study may also occur in other loci, especially oncogene or tumor suppressor genes sites, and may play a role in inducing malignancies in individuals exposed to this or a similar agent.
In epidemiological studies where exposure to complex mixtures of pesticides or combinations including malathion was found, all results found positive associations with genotoxic effects77,92,93,99,100 and carcinogenic effect87. Investigations of pesticides focused on the potential effects of these substances on an individual basis aim to facilitate the analysis and direct public policies. However, although multiple exposures hinder the assessment of the relationships between pesticides and their mutagenic or carcinogenic effect, they more accurately reflect how these compounds are used87.
Among the manuscripts that sought to investigate correlations between malathion exposure and development of different types of cancers, five of them did not find significant positive associations in cases of non-Hodgkin’s lymphoma (NHL)79,91, multiple myeloma82, prostate cancer91, soft tissue sarcoma86, combined lymphatic-hematopoietic cancers, leukemia, lung, colon and rectum, kidney and bladder cancer, and melanoma91. Another three found significant inverse associations between the use of organophosphate and the appearance of NHL78,84 and pancreatic cancer90.
The other half of them obtained positive associations at statistically significant levels for thyroid cancer78, ovarian cancer in menopausal women78, prostate cancer80,88 and its aggressive type83, breast cancer81, NHL94,98 and between body mass index and cancer colonization among men who used malathion89.
In Chile, about 33 years after the first malathion spraying on the city of Arica, Cabello et al.81 conducted a case-control study with women living in the city and women from Iquique, a control city where spraying had never occurred. The authors observed that those with the most extended exposure to malathion were 5.7 times more likely to be diagnosed with breast cancer, besides 30.5% of the cases of metastases found in the exposed group, compared to 16% in the never exposed group (p<0.05). The study concluded that the increased breast cancer mortality rate in the city of Arica has a significant correlation with exposure to malathion sprayed for more than three decades.
Of all the studies included in this systematic review, only one was carried out in Brazil58, which evidences the lack of research regarding the carcinogenic and genotoxic effects of malathion in the country. On the other hand, the use of pesticides, especially in developing countries, has grown over the years, making it necessary to carry out further studies on occupational and environmental exposure to these pesticides103.
The Brazilian Association of Collective Health (ABRASCO) has produced a Technical Note11 warning about products such as malathion, among others, currently used in vector control of arboviruses, since the real damages caused to the environment and human health have not yet been adequately studied or revealed to vulnerable populations, including Public Health workers. Its harmful effects have been disregarded both in the aggravation of viruses and in the emergence of other pathologies such as allergies, immunotoxicity, cancer, hormonal disorders, neurotoxicity, among others11.
CONCLUSION
This systematic returned results that evidenced the mutagenic effect of malathion used as a commercial formulation, that is, containing its analogs malaoxon and isomalathion, and its ability to promote changes in DNA in vivo. Thus, neoplastic processes can be triggered both in animals and in exposed humans once such changes can reach regions of oncogenes or tumor suppressors in the DNA.
The results of the in vitro studies in both animal and human cell cultures exposed to malathion showed DNA damage, chromosomal alterations, sister chromatid exchanges and micronuclei. In vivo experimental studies have shown sufficient evidence regarding the potential of the pesticide both in inducing genetic damage and inducing neoplasms in mammals. Epidemiological studies have shown statistically significant positive associations for thyroid, breast, and ovarian cancer in menopausal women.
The carcinogenic effect of this pesticide and its implications on the environment and humans should be considered, particularly in the context of arbovirus control.
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