Cigarette Smoke Exposure Effects on Male Offspring

Female Balb/c mice (6 weeks, n = 40, Animal Resources Centre, Perth, Australia) were housed at 20±2°C and maintained on a 12∶12 hour light/dark cycle (lights on 06∶00 h). After acclimatization, the mice were divided into 2 groups with equal body weight (BW), the sham exposed (control, n = 20) and cigarette smoke exposed (SE, n = 20) groups. Animals in the SE group were placed inside a perspex chamber (18 liters) filled up with the smoke produced by two cigarettes (Winfield Red, 16 mg or less of tar, 1.2 mg or less of nicotine and 15 mg or less of CO; Philip Morris, Melbourne, Australia) a time with a 5-minute interval between, twice (10∶00 and 15∶30) daily. Control mice were exposed to air in an identical chamber at the same time as previously described [19] (link), [20] (link). Six weeks later, females were mated. Cigarette smoke exposure continued throughout gestation and lactation periods. The sires and offspring were not exposed. Pups were weaned at postnatal day (P)20 and maintained without additional intervention. The dams were culled when the pups weaned.
Male offspring were scarified at P1, P20 (weaning age) and week 13 (W13, mature age) (n = 11–23). They were weighed and anaesthetized with sodium thiopental (0.1 ml/g, i.p., Abbott Australasia PTY. LTD, NSW, Australia). Blood was collected through cardiac puncture and blood glucose was measured (Accu-chek, Roche Diagnostics, Nutley, USA). Mice were killed by decapitation. The left kidneys were fixed with 10% formalin (Sigma, VIC, Australia); the right kidneys were snap frozen in liquid nitrogen and stored at −80°C. Urine was collected from the bladder when available.