Genotoxic effects in occupational exposure to formaldehyde: A study in anatomy and pathology laboratories and formaldehyde-resins production

Formaldehyde (CH₂O), the most simple and reactive of all aldehydes, is a colorless, reactive and readily polymerizing gas at room temperature [1]. It has a pungent suffocating odor that is recognized by most human subjects at concentrations below 1 ppm [2].

According to the Report on Carcinogens, formaldehyde (FA) ranks 25^th in the overall U.S. chemical production, with more than 5 million tons produced each year [3]. FA annual production rises up to 21 million tons worldwide and it has increased in China, for example, in the recent years, with 7,5 million tons produced in 2007. Given its economic importance and widespread use, many people are exposed to FA environmentally and/or occupationally [4]. According to the International Information System on Occupational Exposure to Carcinogens (CAREX), in the period between 1990 and 1993, 36,000 workers were occupationally exposed to FA in Portugal [5].

Occupational exposure involves not only workers in direct production of FA and products containing it, but also in industries utilizing these products, such as those related with construction and household [1]. The most extensive use of FA is in production of resins with urea, phenol and melamine, and also polyacetal resins. These products are used as adhesives in manufacture of particle-board, plywood, furniture and other wood products [2]. FA is also used in cosmetics composition and has an important application as a disinfectant and preservative, reason why relevant workplace exposure may also occur in pathology and anatomy laboratories and in mortuaries [1, 2, 6].

Human studies have shown that chronic exposure to FA by inhalation is associated with eye, nose and throat irritation [7]. Mostly important, several studies report a carcinogenic effect in humans after chronic exposure to FA, in particular an increased risk for nasopharyngeal cancer [8‐12]. Since 2006, International Agency for Research on Cancer (IARC) classifies FA as carcinogenic to humans (Group 1), based on sufficient evidence in humans and in experimental animals [2]. IARC also concluded that there is a "strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde".

With actual scientific evidence we can conclude that, regarding to risk estimation, local toxic effects at site of first contact seem to be the most relevant health effects [2, 7, 13].

Manifold in vitro studies clearly indicated that FA can induce genotoxic effects in proliferating cultured mammalian cells [2]. Furthermore, some in vivo studies detected changes in epithelial cells (oral and nasal) and in peripheral lymphocytes related to FA exposure [13, 14].

Frequency of micronucleus (MN) in buccal and/or nasal mucosa cells is being used to investigate local genotoxicity. According to reports concerning experimental genotoxicity studies, MN are the most sensitive genetic endpoints for detection of FA induced genotoxicity [15]. Thus, MN test with exfoliated cells could be a powerful tool for detection of local genotoxic effects in humans, which is fundamental for hazard identification and risk estimation [13].

MN in peripheral blood lymphocytes has been extensively used to evaluate the presence and extend of chromosome damage in human populations exposed to genotoxic agents. As advantages, this MN test provides a reliable measure of chromosomal breakage and loss at lower cost and more easily than chromosomal aberrations. Moreover, the availability of cytokinesis-block technique eliminates potential background caused by effects on cell division kinetics [16].

The goal of this study is to contribute to the investigation of genotoxic effects in workers occupationally exposed to FA.

As indicated by several studies [6, 21, 22] exposure assessment in present investigation identified that both groups of workers (factory and laboratory) were exposed to high peak FA concentrations.

The importance of this consideration lies in the fact that health effects (cancer) linked to FA exposure are more related with peaks of high concentrations than with long time exposure at low levels [2, 23]. The choice of exposure metric should be based on the most biologically relevant exposure measure in order to diminish misclassification of exposure, thus leading to attenuated exposure-response relationships [24]. Moreover, exposures of short duration (peaks) are of special concern, because they produce an elevated dose rate at target tissues and organs, potentially altering metabolism, overloading protective and repair mechanisms and amplifying tissue responses [24, 25].

Considering this, Pyatt et al. (2008) pointed out, as a limitation in most epidemiological studies, the lack of data about exposure to peak concentrations. Therefore, in those studies, health effects resulting from occupational exposure to FA are associated to exposure exclusively based on time-weighted average concentrations [23]. The only two studies concerning the association between exposure to FA and nasopharyngeal cancer that presented data on exposure to ceiling concentrations obtained higher relative risk values compared with the other studies [1, 12, 26].

Moreover, other groups also suggested ceiling concentrations as the most important exposure metric, when attempting to define the relative risk of myeloid leukaemia in workers exposed to FA [1, 27‐29].

The present results obtained in the laboratories, evidence a difference between the two exposure metrics (0.28 ppm for TWA_{8 h} and 2.52 ppm for ceiling level). These results are in good agreement with previous ones reported by Shaham et al.[30]. A difference of the same order of magnitude was described in 14 pathology laboratories (0.4 ppm for TWA and 2.24 ppm for ceiling level).

Each one of these results would lead to different conclusions about exposure assessment and, consequently, to a different risk assessment and, though, claim our attention for the importance of exposure metric selection. For FA occupational exposure, ceiling concentrations might be a better strategy to evaluate exposure and to develop risk assessment once very high exposures over short periods are missed by TWA_{8 h} method and, in fact, they are important to know the real risk for health [23, 31]. Therefore, as in other investigations [33, 34], it is possible to conclude that when measuring only TWA_{8 h} poor information is obtained, and the method is of less utility to identify processes that should be targeted for controls.

Results indicate macroscopic examination of anatomical specimens FA-preserved as the task involving exposure to the highest values. This occurs because precision and good visibility is required and as a consequence pathologists must lean over the specimen with consequent increase of proximity to FA emission sources. Studies developed by Goyer et al. and Orsière et al. support that proximity to impregnated specimens promotes higher exposure to FA [6, 22].

In factory, the task of collecting samples from resins reactor present the higher exposure, because the reactors are consider the units with larger emissions [6, 32]. Furthermore, the sampling process is still manual in the factory studied.

It is important to notice that the information about exposure determinants, emission sources and exposed workers was only possible because video recording could be performed. This resource gives the opportunity to directly relate performance with exposure [18, 35, 36].

FA genotoxicity is confirmed in a variety of experimental systems ranging from bacteria to rodents in vivo [37]. Although the findings from in vivo animal studies may provide a basis for extrapolation to humans, cytogenetic assays in humans have been conflicting, sometimes with contradictory outcomes [38]. Nevertheless, our results showed a significant increase in MN frequency in epithelial cells and in lymphocytes of exposed individuals compared with controls.

Biological evidence of toxicity on distant-site such as peripheral lymphocytes and bone marrow is still controversial [2, 39]. Some authors have argued that it is biologically implausible for FA to cause leukaemia as FA is unlikely to reach the bone marrow and cause toxicity. Due to its highly reactive nature and rapid metabolism, there is no evidence that it can damage stem and progenitor cells (the target cells for leukemogenesis). Also, there is no credible experimental animal model for FA-induced leukaemia [40, 41]. However, Zhang et al. hypothesize that FA may act on bone marrow directly or, alternatively, may cause leukaemia by damaging the hematopoietic stem or early progenitor cells that are located in the circulating blood or nasal passages, which would then travel to bone marrow and become leukemic stem cells [1, 29]. Nevertheless, our findings are consistent with other previous studies on epithelial cells and also on peripheral lymphocytes [42‐44]. Suruda et al. reported that low-level exposure to FA was associated with cytogenetic changes in buccal epithelial cells and in blood lymphocytes in mortician students [14]. Our results in blood lymphocytes can be an indication that cytogenetic effects can be found in tissues distant from the area of initial contact (nasopharyngeal) and even reach the bone marrow and cause toxicity, supporting the thesis of Zhang and colleagues [1, 29].

A significant positive correlation between MN frequency (both in peripheral blood lymphocytes and in epithelial buccal cells) and the duration of FA exposure (years of employment) was found (Table 4). This indicates that, together with peak contacts, exposure duration also has relevance for the development of health effects. Furthermore, in our study, long-term exposure to high levels of FA was noted particularly in pathology and anatomy laboratory workers (exposure duration mean of 14.5 years), fact that may at least contribute to explain the higher frequency of MN in peripheral blood lymphocytes in this group when compared to the factory group (Table 4). Regarding the influence of age, a positive correlation was found with MN frequency in peripheral blood lymphocytes (Table 5). MN frequencies tend to rise with age because of the progressive increase in spontaneous chromosome instability and the loss of efficiency in DNA repair mechanisms, which may result in accumulation of genetic lesions with increasing age [22, 45]. On the other hand, for MN frequency in epithelial buccal cells, a negative correlation was found (Table 5). This can possibly be explained by the fact that cells of buccal mucosa have a steady and rapid turnover, and therefore accumulation of genotoxic effects becomes difficult [13].

No significant differences were obtained in MN frequencies between women and men (both in peripheral blood lymphocytes and epithelial buccal cells). However, in other studies an increase in MN frequencies in women was found. Current knowledge on the effect of gender on genetic damage determines a 1.5-fold greater MN frequency in females than in males [19, 45], witch can be explained by preferential aneugenic events involving the X-chromossome. Surralés et al. reported an excessive overrepresentation of this chromosome in micronucleic lymphocytes cultured from women [46].

Tobacco smoke contains a high number of mutagenic and carcinogenic substances and is causally linked to an elevated incidence of several forms of cancers [47]. Hence, smoking is an important variable to consider in biomonitoring studies and, particularly in this study since FA is present in tobacco smoke [2]. The effect of tobacco smoking on MN frequency in human cells has been object of study. In most reports the results were unexpected, as in many instance smokers had lower frequencies of MN than non-smokers [22, 48]. In the present study no significant differences were found in MN (peripheral blood lymphocytes and epithelial buccal cells) between smokers and non-smokers. These findings are similar to results obtained in the study of Bonassi et al., [48]. These authors recommend that quantitative data about smoking habit should be collected because the sub-group of heavy smokers (≥30 cigarettes per day) can influence the results. For notice, the questionnaire results of this study revealed no heavy smokers in these workers groups.

In conclusion, the population studied is exposed to high ceiling concentrations (peaks) of FA with a long-term exposure. These two aspects, cumulatively, can be the cause for the increase in MN frequencies in lymphocytes and in epithelial buccal cells.

Results obtained suggest that preventive and protective measures must be applied in order to reduce occupational exposure to this chemical agent in these two occupational settings and, subsequently, to prevent adverse effects on workers health.