In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P <.05) and by 82% within 2 hr (P <.05), accompanied by increases of mitochondrial 4-hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P <.05) at the 6- to 24-hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P <.05), concomitant with activation of caspase 3 (52% at 12 hr, P <.05). Pretreatment with N-acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol-mediated apoptotic death of fetal cortical neurons.

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Aims/hypothesis This randomised controlled trial was performed in India and the UK in people with prediabetes to study whether mobile phone short message service (SMS) text messages can be used to motivate and educate people to follow lifestyle modifications, to prevent type 2 diabetes. Methods The study was performed in people with prediabetes (n = 2062; control: n = 1031; intervention: n = 1031) defined by HbA 1c ≥42 and ≤47 mmol/mol (≥6.0% and ≤6.4%). Participants were recruited from public and private sector organisations in India (men and women aged 35-55 years) and by the National Health Service (NHS) Health Checks programme in the UK (aged 40-74 years without pre-existing diabetes, cardiovascular disease or kidney disease). Allocation to the study groups was performed using a computergenerated sequence (1:1) in India and by stratified randomisation in permuted blocks in the UK. Investigators in both countries remained blinded throughout the study period. All participants received advice on a healthy lifestyle at baseline. The intervention group in addition received supportive text messages using mobile phone SMS messages 2-3 times per week. Participants were assessed at baseline and at 6, 12 and 24 months. The primary outcome was conversion to type 2 diabetes and secondary outcomes included anthropometry, biochemistry, dietary and physical activity changes, blood pressure and quality of life. Results At the 2 year follow-up (n = 2062; control: n = 1031; intervention: n = 1031), in the intention-to-treat population the HR for development of type 2 diabetes calculated using a discrete-time proportional hazards model was 0.89 (95% CI 0.74, 1.07; p = 0.22). There were no significant differences in the secondary outcomes. Conclusions/interpretation This trial in two countries with varied ethnic and cultural backgrounds showed no significant reduction in the progression to diabetes in 2 years by lifestyle modification using SMS messaging. Trial registration The primary study was registered on www.ClinicalTrials.gov (India, NCT01570946; UK, NCT01795833).

These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.