**Genotoxicity biomarkers**

For all genotoxicity biomarkers under study, workers exposed to FA had significantly higher mean values than the controls (Table 3).

In peripheral blood lymphocytes, significant differences (Mann-Whitney test, p<0.001) were observed between subjects exposed and non-exposed to FA, namely in mean MN (respectively, 3.96±0.525 *vs* 0.81±0.172), NPB (3.04±0.523 *vs* 0.18±0.056), and NBUD (0.98±0.273 *vs* 0.07±0.028). In buccal mucosa cells, the MN mean was also significantly higher (p=0.002) in exposed subjects (0.96±0.277) than in controls (0.16±0.058).


Table 3. Descriptive statistics of MN in lymphocytes and buccal cells, NPB and NBUD means in the studied population (mean ± mean standard error, range and p-value of Mann-Whitney test)

Discriminating by occupation, technologists group mean of MN in lymphocytes was 3.76±0.647; in NBP was 2.62±0.629; in NBUD was 1.09±0.401 and in MN in BM was 1.18±0.406. In pathologists, the means were 5.00±1.243; 3.75±1.467; 0.33±0.188 and in MN in BM was 0.58±0.434, respectively.

The odds ratios indicate an increased risk for the presence of biomarkers in those exposed to FA, compared to non-exposed (Table 4) and they were all significant (p<0.001).


Table 4. Results of binary logistic regression concerning the association between FA and genotoxicity biomarkers, as evaluated by the odds ratio (OR).

For all genotoxicity biomarkers under study, workers exposed to FA had significantly

In peripheral blood lymphocytes, significant differences (Mann-Whitney test, p<0.001) were observed between subjects exposed and non-exposed to FA, namely in mean MN (respectively, 3.96±0.525 *vs* 0.81±0.172), NPB (3.04±0.523 *vs* 0.18±0.056), and NBUD (0.98±0.273 *vs* 0.07±0.028). In buccal mucosa cells, the MN mean was also significantly

higher (p=0.002) in exposed subjects (0.96±0.277) than in controls (0.16±0.058).

Mean, NPB±S.E. (range)

**Controls** 0.81±0.172 (0-7) 0.18±0.056 (0-3) 0.07±0.028 (0-1) 0.16±0.058 (0-2) **Exposed** 3.96±0.525 (0-14) 3.04±0.523 (0-15) 0.98±0.273 (0-13) 0.96±0.277 (0-9) **p-value** <0.001 <0.001 <0.001 0.002 Table 3. Descriptive statistics of MN in lymphocytes and buccal cells, NPB and NBUD means in the studied population (mean ± mean standard error, range and p-value of Mann-

Discriminating by occupation, technologists group mean of MN in lymphocytes was 3.76±0.647; in NBP was 2.62±0.629; in NBUD was 1.09±0.401 and in MN in BM was 1.18±0.406. In pathologists, the means were 5.00±1.243; 3.75±1.467; 0.33±0.188 and in MN in

The odds ratios indicate an increased risk for the presence of biomarkers in those exposed to

**MN lymphocytes** 9.665 3.81-24.52 <0.001

Table 4. Results of binary logistic regression concerning the association between FA and

genotoxicity biomarkers, as evaluated by the odds ratio (OR).

**NPB** 11.97 4.59-31.20 <0.001 **NBUD** 9.631 3.12-29.70 <0.001 **MN buccal cells** 3.990 1.38-11.58 0.011

FA, compared to non-exposed (Table 4) and they were all significant (p<0.001).

Table 2. FA ceiling values (ppm) by tasks in the macroscopy room.

**Genotoxicity biomarkers** 

Whitney test)

BM was 0.58±0.434, respectively.

higher mean values than the controls (Table 3).

Mean, MN lymphocytes ±S.E. (range)

Macroscopic specimen's exam 2.93 Disposal of specimen and used solutions 0.95

Tasks Ceiling values

Mean, NBUD±S.E. (range)

OR CI 95% p-value

Jar filling 2.51 Specimen wash 2.28 Biopsy exam 1.91

(ppm)

Mean, MN buccal cells ±S.E. (range)

Regarding the impact of the duration of exposure to FA, the mean values of MN in lymphocytes and in buccal cells tended to increase with years of exposure (Table 5) but the association was not statistically significant.


Table 5. Descriptive statistics according to years of exposure to formaldehyde of MN in lymphocytes and buccal cells, NPB, and NBUD means in the two groups (mean ± mean standard error, range)

Age and gender are considered the most important demographic variables affecting the MN index. However, Table 6 shows that the mean of all the genotoxicity biomarkers did not differ between men and women within the exposed and the controls (p> 0.05).


Table 6. Descriptive statistics by gender of MN in lymphocytes and buccal cells, NPB, and NBUD means in the two groups (mean ± mean standard error, range)

In order to examine the effect of age, exposed and non-exposed individuals were stratified by age groups: 20-30, 31-40, and ≥ 41 years old (Table 7). There was no consistent trend regarding the variation of biomarkers with age, the only exception being the MN in lymphocytes in the exposed group (Kruskal-Wallis, p= 0.006), where the higher means where found in the older group. According to Mann-Whitney test, there is a statistical significant result between the elder and the older group (20–30 and > 41 years old, p= 0.02), however the comparison between 20-30 and 31–40 groups (p= 0.262) and 30–40 and > 41 groups (p= 0.065) did not reach statistical significance.

Genotoxicity Biomarkers: Application in Histopathology Laboratories 147

Long exposures to FA, as those to which some workers are subjected for occupational reasons, are suspected to be associated with genotoxic effects that can be evaluated by biomarkers (Conaway et al., 1996; IARC, 2006; Viegas & Prista, 2007; Zhang et al., 2009). In this study the results suggest that workers in histopathology laboratories are exposed to FA levels that exceed recommended exposure limits. Macroscopic specimens' exam, in particular, is the task that involves higher exposure, because it requires a greater proximity to anatomical species impregnated with FA, as supported by the studies of Goyer et al.

A statistically significant association was found between FA exposure and biomarkers of genotoxicity, namely MN in lymphocytes, NPB, NBUD and MN in buccal cells. Chromosome damage and effects upon lymphocytes arise because FA escapes from sites of direct contact, such as the mouth, originating nuclear alterations in the lymphocytes of those exposed (He & Jin, 1998; IARC, 2006; Orsière et al. 2006; Ye et al., 2005). Our results thus corroborate previous reports (Ye et al., 2005) that lymphocytes can be damaged by long term FA exposure. Moreover, the changes in peripheral lymphocytes indicate that the cytogenetic effects triggered by FA can reach tissues faraway from the site of initial contact (Suruda et al., 1993). Long term exposures to high concentrations of FA indeed appear to have a potential for DNA damage; these effects were well demonstrated in experimental studies with animals, local genotoxic effects following FA exposure, namely DNA-protein cross

In humans, FA exposure is associated with an increase in the frequency of MN in buccal epithelium cells (Burgaz et al., 2002; Speit et al., 2006, 2007b), as corroborated by the results

Suruda el al. (1993) claim that although changes in oral and nasal epithelial cells and peripheral blood cells do not indicate a direct mechanism leading to carcinogenesis, they present evidence that DNA alteration took place. It thus appears reasonable to conclude that FA is a cancer risk factor for those who are occupationally exposed in histopathology

MN and NPB measured in lymphocytes had higher means in pathologists compared with technologists. This result can be explained by the exposure to higher concentrations of pathologists that perform macroscopic exam. Also this chemical mode of action is more

In epidemiological studies, it is important to evaluate the role played by common confounding factors, such as gender, age, smoking and alcohol consumption, upon the association between disease and exposure (Bonassi et al., 2001; Fenech at al., 1999). Concerning gender, studies realized by Fenech et al. (1999) and Wojda et al. (2007) reported that biomarker frequencies were greater in females than in males by a factor of 1.2 to 1.6 depending on the age group. With the exception of MN in the buccal cells of controls, the results presented here point to females having higher frequencies than males in all genotoxicity biomarkers, although the differences usually lacked statistical significance. Such trend is concordant with previous studies that reported higher MN frequency in lymphocytes in females and a slightly higher MN frequency in buccal cells in males (Holland et al., 2008) and that can be explained by preferential aneugenic events involving

related with the concentration than with time of exposure expressed by TWA results.

**2.2.5 Discussion** 

presented here.

laboratories (IARC, 2006).

(2004) and Orsière et al. (2006).

links and chromosome damage (IARC, 2006).


Table 7. Age effects on descriptive statistics of MN in lymphocytes and buccal cells, NPB and NBUD means in the studied population (mean ± mean standard error, range).

The interaction between age and gender in determining the frequencies of genotoxicity biomarkers was investigated and found to be significant only for MN in lymphocytes in exposed subjects (Kruskal-Wallis, p=0.04). In general the MN tended to be more frequent in the > 41 years old category in both genders; however women had the higher means.

Regarding smoking habits, a non-parametric analysis rejected the null hypothesis that biomarkers are the same for the four categories (control smokers and non-smokers, exposed smokers and non-smokers) (Kruskall-Wallis, p<0.001). However, the analysis of the interactions between FA exposure and tobacco smoke between exposed and controls (Mann-Whitney test) showed that FA exposure, rather than tobacco, has a preponderant effect upon the determination of biomarker frequencies. In the control group, non-smokers had slightly higher MN means in buccal cells in comparison with smokers; although the result did not reach statistical significance (Mann-Whitney, p> 0.05).

As for alcohol consumption, because uptake reported in enquires may differ considerably from real consumption, all consumers were gathered into a single entity, in contrast with non-consumers. Nevertheless, no one acknowledged having "heavy drink habits" in the questionnaires.

Overall, biomarkers in controls exhibited higher mean frequencies among alcohol consumers than among non-consumers. Among those exposed, however, mean frequencies were slightly lower among drinkers, suggesting that exposure was the major predominant factor in determining the high biomarker frequencies of those who are exposed. Differences between drinkers and non-drinkers were not statistically significant, to the exception of MN in lymphocytes in controls (Mann-Whitney, p=0.011), where drinkers have higher means. The interaction between alcohol consumption and smoking habits was statistically significant (Kruskal-Wallis, p=0.043), as subjects that do not smoke and do not drink tend to have lower frequencies of MN in buccal cells than those who drink and smoke, with a gradient of frequencies in between.

### **2.2.5 Discussion**

146 Biomarker

**Mean NPB± S.E. (range)**

3.56±0.926 (0-10)

1.20±0.467 (0-4)

3.00±0.879 (0-15)

0.14±0.071 (0-2)

0.20±0.099 (0-3)

0.21±0.155 (0-2)

Table 7. Age effects on descriptive statistics of MN in lymphocytes and buccal cells, NPB and NBUD means in the studied population (mean ± mean standard error, range).

the > 41 years old category in both genders; however women had the higher means.

The interaction between age and gender in determining the frequencies of genotoxicity biomarkers was investigated and found to be significant only for MN in lymphocytes in exposed subjects (Kruskal-Wallis, p=0.04). In general the MN tended to be more frequent in

Regarding smoking habits, a non-parametric analysis rejected the null hypothesis that biomarkers are the same for the four categories (control smokers and non-smokers, exposed smokers and non-smokers) (Kruskall-Wallis, p<0.001). However, the analysis of the interactions between FA exposure and tobacco smoke between exposed and controls (Mann-Whitney test) showed that FA exposure, rather than tobacco, has a preponderant effect upon the determination of biomarker frequencies. In the control group, non-smokers had slightly higher MN means in buccal cells in comparison with smokers; although the result did not

As for alcohol consumption, because uptake reported in enquires may differ considerably from real consumption, all consumers were gathered into a single entity, in contrast with non-consumers. Nevertheless, no one acknowledged having "heavy drink habits" in the

Overall, biomarkers in controls exhibited higher mean frequencies among alcohol consumers than among non-consumers. Among those exposed, however, mean frequencies were slightly lower among drinkers, suggesting that exposure was the major predominant factor in determining the high biomarker frequencies of those who are exposed. Differences between drinkers and non-drinkers were not statistically significant, to the exception of MN in lymphocytes in controls (Mann-Whitney, p=0.011), where drinkers have higher means. The interaction between alcohol consumption and smoking habits was statistically significant (Kruskal-Wallis, p=0.043), as subjects that do not smoke and do not drink tend to have lower frequencies of MN in buccal cells than those who drink and smoke, with a

**Mean NBUD± S.E. (range)** 

1.63±0.816 (0-13)

0.50±0.224 (880-2)

0.69±0.234 (0-5)

0.08±0.047 (0-1)

0.06±0.040 (0-1)

0.07±0.71 (0-1)

**Mean MN buccal cells ± S.E. (range)** 

0.75±0.470 (0-6)

0.40±0.221 (0-2)

1.46±0.503 (0-9)

0.19±0.96 (0-2)

0.14±0.83 (0-2)

0.14±0.143 (0-2)

**Mean MN lymphocytes ± S.E. (range)**

(0-8)

(0-8)

(0-15)

(0-3)

(0-7)

(0-6)

**20-30 18** 2.19±0.526

**31-40 11** 3.00±0.775

**>41 27** 5.54±0.876

**20-30 36** 0.47±0.157

**31-40 35** 1.14±0.326

**>41 14** 0.86±0.501

reach statistical significance (Mann-Whitney, p> 0.05).

**Groups Age N** 

**Exposed** 

**Controls** 

questionnaires.

gradient of frequencies in between.

Long exposures to FA, as those to which some workers are subjected for occupational reasons, are suspected to be associated with genotoxic effects that can be evaluated by biomarkers (Conaway et al., 1996; IARC, 2006; Viegas & Prista, 2007; Zhang et al., 2009). In this study the results suggest that workers in histopathology laboratories are exposed to FA levels that exceed recommended exposure limits. Macroscopic specimens' exam, in particular, is the task that involves higher exposure, because it requires a greater proximity to anatomical species impregnated with FA, as supported by the studies of Goyer et al. (2004) and Orsière et al. (2006).

A statistically significant association was found between FA exposure and biomarkers of genotoxicity, namely MN in lymphocytes, NPB, NBUD and MN in buccal cells. Chromosome damage and effects upon lymphocytes arise because FA escapes from sites of direct contact, such as the mouth, originating nuclear alterations in the lymphocytes of those exposed (He & Jin, 1998; IARC, 2006; Orsière et al. 2006; Ye et al., 2005). Our results thus corroborate previous reports (Ye et al., 2005) that lymphocytes can be damaged by long term FA exposure. Moreover, the changes in peripheral lymphocytes indicate that the cytogenetic effects triggered by FA can reach tissues faraway from the site of initial contact (Suruda et al., 1993). Long term exposures to high concentrations of FA indeed appear to have a potential for DNA damage; these effects were well demonstrated in experimental studies with animals, local genotoxic effects following FA exposure, namely DNA-protein cross links and chromosome damage (IARC, 2006).

In humans, FA exposure is associated with an increase in the frequency of MN in buccal epithelium cells (Burgaz et al., 2002; Speit et al., 2006, 2007b), as corroborated by the results presented here.

Suruda el al. (1993) claim that although changes in oral and nasal epithelial cells and peripheral blood cells do not indicate a direct mechanism leading to carcinogenesis, they present evidence that DNA alteration took place. It thus appears reasonable to conclude that FA is a cancer risk factor for those who are occupationally exposed in histopathology laboratories (IARC, 2006).

MN and NPB measured in lymphocytes had higher means in pathologists compared with technologists. This result can be explained by the exposure to higher concentrations of pathologists that perform macroscopic exam. Also this chemical mode of action is more related with the concentration than with time of exposure expressed by TWA results.

In epidemiological studies, it is important to evaluate the role played by common confounding factors, such as gender, age, smoking and alcohol consumption, upon the association between disease and exposure (Bonassi et al., 2001; Fenech at al., 1999). Concerning gender, studies realized by Fenech et al. (1999) and Wojda et al. (2007) reported that biomarker frequencies were greater in females than in males by a factor of 1.2 to 1.6 depending on the age group. With the exception of MN in the buccal cells of controls, the results presented here point to females having higher frequencies than males in all genotoxicity biomarkers, although the differences usually lacked statistical significance. Such trend is concordant with previous studies that reported higher MN frequency in lymphocytes in females and a slightly higher MN frequency in buccal cells in males (Holland et al., 2008) and that can be explained by preferential aneugenic events involving

Genotoxicity Biomarkers: Application in Histopathology Laboratories 149

concluded that neither alcohol nor smoking, alone, increase MN frequency in buccal cells, but a combination of both resulted in a significant elevation in micronucleated cells in the buccal mucosa. However, the synergism between alcohol consumption and tobacco has not been observed to act upon all biomarkers and, in several studies of lifestyle factors, it was difficult to differentiate the effect of alcohol from that of smoking (Holland et al., 2008).

The CBMN assay is a simple, practical, low cost screening technique that can be used for clinical prevention and management of workers subjected to occupational carcinogenic risks, namely exposure to a genotoxic agent such as FA. The results obtained in this study provide unequivocal evidence of association between occupational exposure to formaldehyde in histopathology laboratory workers and the presence of nuclear changes.

Given these results, preventive actions must prioritize safety conditions for those who perform macroscopic exams. In general, exposure reduction to FA in this occupational setting may be achieved by the use of adequate local exhaust ventilation and by keeping

Another important application of biological monitoring, besides exposure assessment, is the use of biomarkers, at either individual or group level, for the correct interpretation of doubtful clinical tests. These are usually performed as part of occupational health surveillance program when exposure assessment data are unavailable or are deemed unreliable. Health surveillance is the periodical assessment of the workers' health status by clinical, biochemical, imaging or instrumental testing to detect any clinically relevant, occupation-dependent change of the single worker's health. Biomarkers are usually more specific and sensitive than most clinical tests and may be more effective, therefore, for assessing a causal relationship between health impairment and chemical exposure when a

Experience in biological monitoring gained in the occupational setting has often been applied to assess (the effects of) human exposure to chemicals in the general environment. The use of biological fluids/tissues for the assessment of human exposure, effect or susceptibility to chemicals in the workplace represents, together with the underlying data (e.g. personal exposure and biological monitoring measurements, media-specific residue measurements, product use and time-activity information), a critical component of the occupational risk assessment process, a rapidly advancing science (Manno et al., 2010).

Au et al. (1998), advise to put more emphasis on monitoring populations which are known to be exposed to hazardous environmental contaminant and on providing reliable health risk evaluation. The information can also be used to support regulations on protection of the

This work was supported by Autoridade Portuguesa para as Condições de Trabalho (ACT – Portuguese Authority for Work Conditions). http://www.act.gov.pt/. Project reference:

biological specimen containers closed during the macroscopic exam.

change is first detected in exposed workers (Manno et al., 2010).

**3. Conclusion** 

environment.

075MNA/06.

**4. Acknowledgment**

the X-chromosome. A possible explanation is the micronucleation of the X chromosome, which has been shown to occur in lymphocytes in females, both *in vitro* and *in vivo*, and that can be accounted for by the presence of two X chromosomes. This finding might explain the preferential micronucleation of the inactive X (Catalán et al., 1998, 2000a, 2000b).

Aging in humans appears to be associated with genomic instability. Cytogenetically, ageing is associated with a number of gross cellular changes, including altered size and morphology, genomic instability and changes in expression and proliferation (Bolognesi et al., 1999; Zietkiewicz et al., 2009). It has been shown that a higher MN frequency is directly associated with decreased efficiency of DNA repair and increased genome instability (Kirsch-Volders et al., 2006; Orsière et al., 2006). The data has shown a significant increase of MN in lymphocytes in the exposed group. This can be explained in light of genomic instability, understood as an increased amount of mutations and/or chromosomal aberrations that cytogenetically translate into a greater frequency of changes in chromosome number and/or structure and in the formation of micronuclei (Zietkiewicz et al., 2009). The involvement of micronucleation in age-related chromosome loss has been supported by several studies showing that the rate of MN formation increases with age, especially in women (Catalán et al., 1998). This study provides evidence that age and gender interact to determine the frequency of MN in the lymphocytes of exposed subjects. The higher incidence of MN in both genders is more manifest in older age groups and the effect of gender becomes more pronounced as age increases. Several reports link this observation to an elevated loss of X chromosomes (Battershill et al., 2008).

Tobacco smoke has been epidemiologically associated to a higher risk of cancer development, especially in the oral cavity, larynx, and lungs, as these are places of direct contact with the carcinogenic tobacco's compounds. In this study, smoking habits did not influence the frequency of the genotoxicity biomarkers; moreover, the frequencies of MN in buccal cells were unexpectedly higher in exposed non-smokers than in exposed smokers, though the difference was not statistically significant. In most reports, the results about the effect of tobacco upon the frequency of MN in human lymphocytes were negative as in many instances smokers had lower MN frequencies than non-smokers (Bonassi et al., 2003). In the current study, the analysis of the interaction between FA exposure and smoking habits indicates that exposure is preponderant in determining the frequency of biomarkers. Nevertheless, the effect of smoking upon biomarkers remains controversial. Some studies reported an increased frequency of MN in lymphocytes, NPB, and NBUD as a consequence of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Still in this study no associations were observed between tobacco and nuclear abnormalities (El-Zein et al., 2006, 2008).

As for alcohol consumption, it did not appear to influence the frequency of genotoxicity biomarkers in study, to the exception of MN in lymphocytes in controls (Mann-Whitney, p=0.011), with drinkers having higher means. Alcohol is definitely a recognized genotoxic agent, being cited as able to potentiate the development of carcinogenic lesions (Ramirez & Saldanha, 2002). In our study, drinkers in the control group had higher mean frequencies of all biomarkers than non-drinkers, but the differences were only significant for MN in lymphocytes. Stich and Rosin (1983) study of alcoholic individuals, reported absence of significant differences concerning MN frequencies in buccal cells. That is important to corroborate our result, because of the lack of "heavy drinkers" in our study. The same study concluded that neither alcohol nor smoking, alone, increase MN frequency in buccal cells, but a combination of both resulted in a significant elevation in micronucleated cells in the buccal mucosa. However, the synergism between alcohol consumption and tobacco has not been observed to act upon all biomarkers and, in several studies of lifestyle factors, it was difficult to differentiate the effect of alcohol from that of smoking (Holland et al., 2008).

The CBMN assay is a simple, practical, low cost screening technique that can be used for clinical prevention and management of workers subjected to occupational carcinogenic risks, namely exposure to a genotoxic agent such as FA. The results obtained in this study provide unequivocal evidence of association between occupational exposure to formaldehyde in histopathology laboratory workers and the presence of nuclear changes.

Given these results, preventive actions must prioritize safety conditions for those who perform macroscopic exams. In general, exposure reduction to FA in this occupational setting may be achieved by the use of adequate local exhaust ventilation and by keeping biological specimen containers closed during the macroscopic exam.
