Evaluation of genome damage in subjects occupationally exposed to possible carcinogens. D. Želježi, M. Mladini, N. Kopjar, A. Hursidic Radulovic

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1 Evaluation of genome damage in subjects occupationally exposed to possible carcinogens D. Želježi, M. Mladini, N. Kopjar, A. Hursidic Radulovic

2 WHO Cancer prevention: occupational exposures primary prevention - avoid exposure to carcinogens elimination substitution insulation (encapsulation, automation)

3 WHO Cancer prevention: occupational exposures and secondary prevention or minimize exposure level safe procedure shortening exposure time personal protective equipment (PPE) education (alcohol, tobacco, physical activity) preventive medical examinations

4 Poison Control Centre IMI phenylhydrazine animal carcinogen (the induction of lung and blood vessel tumors in mice) or is suspected to cause the Ca in humans: lung, colon, and melanoma ethylene oxide: human carcinogen :leukemia and other hematopoietic Ca, stomach cancer, brain, pancreas dichloromethane: grupa 2 B animal carcinogen, lung, liver, and mammary gland tumors in mice and rats as well as 1,2-dichloroethane, is suspected to increase the risk of brain, pancreatic, lymphatic, hematopoietic, and stomach cancer

5 Micronucleus (MN) assay valuable cytogenetic biomarker of effect, highly correlating with cancer frequencies of the nuclear buds (NBs) and nucleoplasmic bridges (NPBs) In Croatia, for the general population established baseline to 13 ± 2 mikronuklea at 1000 binuclear lymphocytes. Lymphocytes with secondary damage are retained in the blood for up to a year - reflects harmful effects of agents on the genetic material back 6-8 months.

6 Comet assay - to evaluate early effects of exposure on DNA - detecting primary damage to DNA

7 Comet-FISH - comet assay coupled with fluorescent in situ hybridization - to determine the impact of exposure on structural integrity of TP 53 Comet-FISH images of lymphocytes. TP 53, red signal; centromere 17, green signal; counterstained with DAPI counterstain. (a and b) Lymphocytes with both or single TP 53 gene deletions; (c) normal lymphocyte; and (d) fragmented TP 53 gene; bar 10 mm. FISH: fluorescent in situ hybridization; DAPI: 40,6-diamidino-2-phenylindole.

8 What affects the search results Working with pesticides in the orchard / vineyard / flower stand without a mask and protective gloves Working with solvents, paints and protection devices for metal and wood. Intensive work with cleaners and disinfectants based on chlorine Smoking, alcohol Prolonged use of antibiotics Prolonged intake of vitamins Frequent medical tests, ultrasound, CT, MRI) and physical therapy (ultrasound, gs, MGN, Producer) Longer virus, allergies, chronic inflammation Hereditary factors

9 Study population and blood sampling initial exposed population - 42 subjects total exposure 5 h/ d/ 5 d per week through a questionnaire regarding personal medical history and lifestyle reduced to 16-9 females and 7 males - smokers (4)& nonsmokers (12) average age years (range 25 55) following 8 months of strict use of PPE - second blood sampling

10 Table 1. Number of micronuclei, nuclear buds and nucleoplasmic bridges in lymphocytes of pharmaceutical workers exposed to PHZ, EtO, DCM, DCE. Samplin g time Irregular PPE use Strict PPE use Subgroup TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Non-smokers (n=12) TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Non-smokers (n=12) Frequency of aberrant chromatin structures in 1000 binuclear lymphocytes MN (±S.D.) NB (±S.D.) NPB (±S.D.) 12.4± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.6* 10.0± ± ±3.9 * 9.8± ± ± ± ± ± ± ± ± ± ±1.1 For each subject 1000 cells were scored per duplicate. PHZ-phenylhdrazyne; EtO-ethylene oxide; DCM- dichloromethane; DCE-1,2-dichloroethane; *P <0.05; versus sampling time 1.

11 Table 2. Results for the alkaline comet assay in leukocytes of pharmaceutical workers exposed to PHZ, EtO, DCM, DCE. Parameters of the alkaline comet assay scored in For each subject Sampling 100 leukocytes 100 cells Subgroup were scored time per Irregular PPE use Strict PPE use TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Nonsmokers (n=12) TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Nonsmokers (n=12) TL (mean±s.d.) % DNA in tail (mean±s.d.) 19.8± ± ± ± ± ± ± ± ± ± ±1.3 * 0.7±0.5 * 12.9±1.0 * 0.5±0.2 * 13.1±1.6 * 1.0±0.8 * 13.2± ±0.2 * 12.9±1.3 * 0.8±0.6 * duplicate. PHZ-phenylhdrazyne; EtO-ethylene oxide; DCM- dichloromethane; DCE-1,2-dichloroethane; *P <0.05; versus sampling time 1.

12 Table 3. Results for comet-fish assay showing fragmentation rate and deletion incidence of TP 53 gene and centromere of chromosome 17 in leukocytes of pharmaceutical workers exposed to PHZ, EtO, DCM, EDC. Samplin g time Irregular PPE use Strict PPE use Subgroup TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Nonsmokers (n=12) TOTAL (n=16) Women (n=9) Men (n=7) Smokers (n=4) Nonsmokers (n=12) % (±S.D.) of cells regarding number of TP 53 signals (red) in comet tail Deletion s % (±S.D.) of cells regarding number of cen 17 signals (green) in comet tail Deletions 75.6± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±6.3 * 18.2±4.7 * 4.2± ± ±3.2 * 3.7± ± ± ± ± ± ± ± ± ± ± ±5.8 * 17.9±3.3 * 4.8±2.6 * 13.9± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±5.1 * 3.0±2.6 * 2.8±3.1 1 both signals were located in the head of the comet (scored duplicate slides with 30 cells); *p<0.05 versus sampling time 1; Green signal is FITC-labelled probe for cen 17, while red signal is Texas Red-labelled probe for TP 53 gene. Column heading with number 2 represents occurrence of both centromere/genes in the tail of the comet, number 1 represent occurrence of only one centromere/gene in the tail, while the other remained in the head; PHZ-phenylhdrazyne; EtO-ethylene oxide; DCM- dichloromethane; DCE-1,2-dichloroethane;

13 To conclude findings speak in favor that strict use of PPE is efficient in minimizing the genotoxic risk from occupational exposure to mixture of known and suspected carcinogens, enforcement of safety procedures led to the decrease of primary DNA damage