SUPPLEMENTARY MATERIAL

Materials and methods

Four pregnant strain H mice, aged 12-14 weeks and weighing on an average ~27 g, were housed as previously described (Balansky et al. 2007, 2009, 2012a, 2012b). Within 12 h after birth, the newborns from one litter (11 mice) started to be exposed to CS. The mice from another litter (8 mice) were left untreated and served as controls of mice exposed to CS early in life. After weaning, the mice from the other two litters (17 mice) were divided by gender and kept in 4 cages, two of males and two of females. At 4 months of age, half males and half females started to be exposed to CS, whereas the remaining mice were left untreated and served as controls of mice exposed to CS in adulthood. Housing and treatments of mice were in accordance with national and institutional guidelines.

Both neonatal mice and 4-month old adult mice were exposed daily to mainstream CS for 4 weeks, using Kentucky 2R4F reference cigarettes (University of Kentucky, Lexington, KY), as described by Balansky et al. (2007). The average concentration of total particulate matter in the exposure chambers was 592 mg/m3. After 4 weeks, the mice were deeply anesthetized with diethyl ether and killed by cervical dislocation. The lungs were subjected to enzymic disgregation (Devereux 1984), and the cell suspensions were divided into aliquots.

The cell cycle stage was assessed by cytofluorimetric cell-cycle analyses (DAKO GalaxyTM Flow Cytometry System, DAKO Cytomation, Glostrup, Denmark), using cells permeabilized in 80% methanol (-20°C) and incubated for 30 min in a solution containing RNase and of the probe propidium iodine (Krishan 1975).

Smears of lung cells from each mouse were stained with May-Grünwald-Giemsa. At least 1000 cells were scored, at a 100x magnification, for evaluating the frequencies of micronuclei and necrotic cells (Fenech 2007).

The remaining aliquots were immediately challenged with the various fluorochromes for the analysis of freshly prepared cells. The fluorescent LysoTrackerTM red DND-99 probe (Molecular Probes, Eugene, OR, USA) was used for evaluating autophagy (Raben et al. 2009). 8-Oxo-dGuo was measured by fluorescein isothiocyanate (FITC)-labeled avidin binding (Struthers et al. 1998). FACS analyses of lipid peroxides used the probe diphenyl-1-pyrenylphosphine (DPPP) (Invitrogen Molecular Probe, Milan, Italy) (Takahashi et al. 2001). The intracellular H2O2 content was evaluated by using the lipophilic probe dihydrorodamine DHR123 (Rothe and Valet 1990). For evaluating the mitochondrial mass, the cells were incubated with 10-nonyl bromide acridine orange (NAO, Invitrogen Molecular Probes) (Limoli et al. 2003). The mitochondrial membrane potential was evaluated by measuring the incorporation of the fluorescent probe rhodamine 123 (R123, Invitrogen Molecular Probes) (Limoli et al. 2003).

The body weights of mice were expressed as means ± SE within each experimental group. The significance of the differences between individual groups was evaluated by Student’s t test for unpaired data. All analytical data were expressed as medians ± 95% CI (confidence intervals) within each experimental group. The significance of the differences between individual groups was evaluated by nonparametric Mann-Whitney test, after having checked the normality of data by Wilcoxon test.

References

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Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2:1084-1104.

Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188-193.

Limoli CL, Giedzinski E, Morgan WF, Swarts SG, Jones GD, Hyun W (2003) Persistent oxidative stress in chromosomally unstable cells. Cancer Res 63:3107-3111.

Raben N, Shea L, Hill V, Plotz P (2009) Monitoring autophagy in lysosomal storage disorders. Methods Enzymol 453:417-449.

Rothe G, Valet G (1990) Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin. J Leukoc Biol 47:440-448.

Struthers L, Patel R, Clark J, Thomas S (1998) Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogues. Anal Biochem 255:20-31.

Takahashi M, Shibata M, Niki E (2001) Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine. Free Radic Biol Med 31:164-174.