24 – the methodology of sister chromatid exchanges

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics—halothane and isoflurane—in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1–10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0.56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37°C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.

Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay

OBJECTIVES: To evaluate genetic damage as the frequency of sister chromatid exchanges and micronuclei in lymphocytes of peripheral blood of operating room personnel exposed to waste anaesthetic gases. METHODS: Occupational exposure was measured with a direct reading instrument. Venous blood samples were drawn from 10 non-smokers working in the operating room and 10 non-smoking controls (matched by age, sex, and smoking habits). Lymphocytes were cultured separately over 72 hours for each assay with standard protocols. At the end of the culture time, the cells were harvested, stained, and coded for blind scoring. The exchanges of DNA material were evaluated by counting the number of sister chromatid exchanges in 30 metaphases per probe or by counting the frequency of micronuclei in 2000 binucleated cells. Also, the mitotic and proliferative indices were measured. RESULTS: The operating room personnel at the hospital were exposed to an 8 hour time weighted average of 12.8 ppm nitrous oxide and 5.3 ppm isoflurane. The mean (SD) frequency of sister chromatid exchanges was significantly higher (10.2 (1.9) v 7.4 (2.4)) in exposed workers than controls (p = 0.036) the proportion of micronuclei (micronuclei/500 binucleated cells) was also higher (8.7 (2.9) v 6.8 (2.5)), but was not significant (p = 0.10). CONCLUSION: Exposure even to trace concentrations of waste anaesthetic gases may cause dose-dependent genetic damage. Concerning the micronuclei test, no clastogenic potential could be detected after average chronic exposure to waste anaesthetic gas. However, an increased frequency of sister chromatid exchanges in human lymphocytes could be detected. Although the measured differences were low, they were comparable with smoking 11-20 cigarettes a day. Due to these findings, the increased proportion of micronuclei and rates of sister chromatid exchanges may be relevant long term and need further investigation.

https://oem.bmj.com/content/oemed/56/7/433.full.pdf

Genetic damage in operating room personnel exposed to isoflurane and nitrous oxide.

Metallurgy has been taken as essential to the development of Chinese civilization. Archaeological study has been particularistic and evolutionary, tied to traditional Chinese historiography, and modern Marxist models of social development. Modern studies suggest that metallurgy emerged independently in a ‘core’ area and then spread to ‘peripheral’ areas by way of political expansion and cultural diffusion over many millennia, and that metallurgy was also homegrown. New excavations suggest: multiple early centers of production; that the Chinese case belongs to a regional context; that native sources of ores were significant; that metals were used in several pre-state level societies.

Metallurgy in Ancient Eastern Asia: Retrospect and Prospects

Sister-chromatid exchange (SCE) analysis was carried out in 67 operating room personnel (anaesthetists M.D.; anaesthesia nurses and anaesthesia unit technicians) exposed to waste anaesthetic gases such as halothane, nitrous oxide and isoflurane and in 50 healthy unexposed controls. The SCE frequencies were increased significantly in operating room personnel as compared to controls. A significant increase in SCEs was found in non-smoking operating room personnel as compared to non-smoking controls. This study supports the existence of an association between occupational exposure to mutagens and an increase in SCEs in lymphocytes.

Sister-chromatid exchanges in operating room personnel

Sister chromatid exchanges (SCE) and sister chromatid exchange points (SCE-points) were counted in lymphocytes in peripheral blood drawn from hospital personnel exposed to anaesthetics as well as from persons not exposed.



A total of 38 healthy persons were investigated, representing female nurse anaesthetists, male physicians practising anaesthesia, female nurses from the intensive care unit, and female secretaries.



The mean SCE number per cell for each person was used as the variable, and the Mann-Whitney U-test was applied to test for differences between groups. The group of secretaries seemed to differ from the other three groups, which appeared identical (P<0.001). Correlation of cigarette smoking and number of SCE could not be demonstrated (r=.255, n = 38).



It was concluded that by this method there was no indication of a mutagen effect of long-term exposure to waste anaesthetic gases such as halothane and nitrous oxide.

H. C. Wulf

Papers

Sister Chromatid Exchanges in Lymphocytes in Operating Room Personnel