Study population and sample selection

Our sample selection is described with the flow chart in Fig. 2. Of the 319 females included, 261 gave blood samples, of which 254 could be used for the comet assay. Nine samples were excluded due to insufficient cell viability. The results presented in this study rely on 245 female samples.

Fig. 2

Flow chart of sample selection

Socio-demographic characteristics associated to DNA damage

Mean age of the 245 females analyzed was 46 years old at enrollment (ranging from 19 to 73 years), 58% of them were of normal BMI (18.5–25 kg/m2). The majority of the females were never smokers (80%) and 45% consumed alcohol occasionally (Table 1).

Table 1 Univariable analyses between socio-demographic characteristics and DNA damage

There was no variation in DNA damage levels with age, BMI, or alcohol consumption. Smoking at the time of sampling was associated with a decrease in DNA damage compared with no smoking history (p = 0.01). Former smoking revealed a tendency of decreased DNA damage compared with no smoking history (p = 0.09). We observed a trend of decreased DNA damage in association with smoking and former smoking (p = 0.01). A tendency of decreased DNA damage was found with an increased quantity of cigarettes smoked per day (p = 0.09) (Table 1), only when current smokers are compared to ex- and never-smokers together.

Occupational exposure associated to DNA damage

Participants undertaking tasks associated with crops were lowly represented, where the most implication was for harvest work with 34 women (14%). The use of herbicides on meadows was not associated with differences in DNA damage levels (p = 0.68). However, a longer duration of use of herbicides on meadows for people undertaking this task show a tendency of higher DNA damage (n = 5, p = 0.05). Cleaning and upkeep of agricultural equipment was not associated with modified DNA damage levels (p = 0.65), but a longer duration of practicing this task showed a tendency to have higher DNA damage levels (n = 18, p = 0.06). Regarding tasks related to livestock, people undertaking milking tended to have higher DNA damage levels (p = 0.16) (Table 2).

Table 2 Univariable linear regression for occupational exposure and DNA damageEnvironmental farmland exposure associated to DNA damage

Farm characteristics were assessed by a separate questionnaire which was filled in by the farm owner, describing what is present on the farm regardless of individual activities, therefore assessing the general working and living environment participants are exposed to.

Increased utilized agricultural area (UAA) overall on the farm was associated with lower levels of DNA damage (p = 0.01).

Regarding specific crops, no modification of DNA damage levels was found in people working on farms where rape, meadows or wheat crops are present (rape p = 0.64, meadows p = 0.93 and wheat p = 0.56 respectively). However, an increased area of rape and meadows were associated with reduced levels of DNA damage (p = 0.04 and p = 0.03 respectively), there was also a tendency of reduced DNA damage levels with increased area of wheat crops (p = 0.07).

No association was visible with the presence of orchards (p = 0.98), but DNA damage levels tended to be higher with increasing orchard size (p = 0.19).

The presence of flax and pea crops on the farm led to a tendency to lower DNA damage (p = 0.18 and p = 0.15). However, size of these crops was not associated with modified DNA damage levels (p = 0.34 for flax and p = 0.33 for pea).

Concerning livestock, the presence of sheep, cattle, and goats was not associated with modification of DNA damage levels (sheep p = 0.62, cattle p = 0.82, goats p = 0.53). However, increasing number of sheep on the farm was associated with reduced DNA damage levels (p = 0.04), and there was also a tendency to reduced DNA damage levels with the number of cattle and goats (p = 0.17 and p = 0.06, respectively) (Table 3).

Table 3 Farm characteristics and univariable association with DNA damage score

Having pigs present on farms was associated with a higher amount of DNA damage (p = 0.04), though the number of pigs was not associated with modified DNA damage levels (p = 0.95).

There was a tendency to lower DNA damage when poultry were present on the farm (p = 0.15), but without link with the number of animals (p = 0.56).

Multivariable analysis of parameters associated with DNA damage

We ran a multivariate analysis with the parameters with which an association or tendency with modified DNA damage levels, either increase of decrease, was detected. Current and former smoking remained associated with lower DNA damage levels (p = 0.0008 with β = −0.80 and p = 0.03 with β = −0.48 respectively), as well as the trend associating smoking with decreased DNA damage (p = 0.0009).

No direct occupational exposure remained associated with DNA damage in the multivariate analysis.

Regarding exposures to the farming environment, pig farming was associated with higher DNA damage, (p = 0.01) though the presence of poultry farming and the bigger the surface of meadows were associated with lower DNA damage (p = 0.003 and p = 0.04, respectively) (Table 4).

Table 4 Multivariable analysis of exposure and DNA damageAnalysis of parameters associated to DNA damage including HDCs

We replicated our analysis with HDCs included in the comet score.

The relationship between smoking status and reduced DNA damage remained for current smokers, but as a tendency (p = 0.10), and was no longer present for former smokers, nor was the trend of smoking overall (supplementary data table S2).

Concerning occupational exposure, all associations/tendencies remained, although the p values were numerically changed, as could be expected (supplementary table S3).

Concerning farm characteristics, the tendency of lower DNA damage with the presence of flax crops on the farm remained (p = 0.16), while it was lost with peas crops. The utilized agricultural area was no longer associated with decreased DNA damage. The tendency of decreased DNA damage with increasing wheat surface disappeared. Increasing size of rape crops and meadows continued to be associated with lower DNA damage, though only as a tendency for meadows (p = 0.04 and p = 0.14 respectively). The tendency of higher DNA damage with the presence of orchards remained (p = 0.19), as it was with the presence of pigs (p = 0.12), although as a tendency. The presence of poultry however lost its association with DNA damage. Regarding the number of sheep and goats, only the number of sheep continued to be associated with lower DNA damage (p = 0.04) (supplementary data table S4).