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Background: The higher incidence of smoking among alcoholic subjects suggests the presence of common molecular mechanisms underlying nicotine and alcohol use and abuse. However, these mechanisms are largely unknown. By using cultured fetal mouse cortical neurons as a model system, we sought to identify genes and pathways that are modulated in the cells by ethanol, nicotine, or both. Methods: Primary cerebral cortical cultures were prepared from the brains of 14-day-old C57BL/6 mouse fetuses and exposed to ethanol (75 mM), nicotine (0.1 mM), or both for 5 consecutive days. A homeostatic pathway-focused microarray consisting of 638 sequence-verified genes was used to measure transcripts differentially regulated by ethanol, nicotine, or both in 5 drug-treated cortical neuron samples and 5 control samples. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was used to verify the mRNA expression levels of genes of interest detected from the microarray experiments. Results: Through a pathway-focused cDNA microarray and balanced experimental design, we identified 65, 111, and 81 significantly regulated genes in the ethanol, nicotine, and ethanol/nicotine-treated neurons, respectively. Of them, the genes of Akt2, Nsg1, Pdgfa, Pfn1, Rbbp7, and Tcfeb were comodulated. The genes differentially expressed in 1 or more treatment groups could be classified into 4 major clusters, with each cluster consisting of genes involved in different biological processes. The platelet-derived growth factor (PDGF) signaling pathway was significantly regulated by all 3 treatments, but by different mechanisms, which may lead to different cellular consequences. Conclusions: Our results indicate that the PDGF pathway represents one of the major biochemical mechanisms in the cellular and molecular responses to each drug in cortical neurons. Finally, we demonstrated that the pathway-focused microarray system used in the present study is a valuable tool for dissecting the mechanisms of complex signaling pathways such as the PDGF pathway.

Previous human and animal studies demonstrate that acute nicotine exposure has complicated influences on the function of the nervous system, which may lead to long-lasting effects on the behavior and physiology of the subject. To determine the genes and pathways that might account for long-term changes after acute nicotine exposure, a pathway-focused oligoarray specifically designed for drug addiction research was used to assess acute nicotine effect on gene expression in the neuron-like SH-SY5Y cells. Our results showed that 295 genes involved in various biological functions were differentially regulated by 1 h of nicotine treatment. Among these genes, the expression changes of 221 were blocked by mecamylamine, indicating that the majority of nicotine-modulated genes were altered through the nicotinic acetylcholine receptors (nAChRs)-mediated signaling process. We further identified 14 biochemical pathways enriched among the nicotine-modulated genes, among which were those involved in neural development/synaptic plasticity, neuronal survival/death, immune response, or cellular metabolism. In the genes significantly regulated by nicotine but blocked by mecamylamine, 13 enriched pathways were detected. Nine of these pathways were shared with those enriched in the genes regulated by nicotine, including neuronal function-related pathways such as glucocorticoid receptor signaling, p38 MAPK signaling, PI3K/AKT signaling, and PTEN signaling, implying that nAChRs play important roles in the regulation of these biological processes. Together, our results not only provide insights into the mechanism underlying the acute response of neuronal cells to nicotine but also provide clues to how acute nicotine exposure exerts long-term effects on the nervous system.

Vitamin K1, K2, and K3 are essential nutrients associated with blood clotting and bone metabolism. Quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] are among the selected enzymes that catalyze reduction of vitamin K to vitamin K hydroquinone. NQO1 catalyzes high affinity reduction of vitamin K3 but has only weak affinity for reduction of vitamin K1 and K2. Vitamin K hydroquinone serves as a cofactor for vitamin K gamma-carboxylase that catalyzes gamma-carboxylation of specific glutamic acid residues in Gla-factors/proteins leading to their activation and participation in blood clotting and bone metabolism. Concomitant with Gla modification, a reduced vitamin K molecule is converted to vitamin K epoxide, which is converted back to vitamin K by the enzyme vitamin K epoxide reductase to complete vitamin K cycle. Vitamin K is also redox cycled. One-electron reduction of vitamin K3 leads to the formation of semiquinone that in the presence of oxygen is oxidized back to vitamin K3. Oxygen is reduced to generate reactive oxygen species (ROS) that causes oxidative stress and cytotoxicity. Vitamin K is used as radiation sensitizer or in mixtures with other chemotherapeutic drugs to treat several types of cancer. ROS generated in redox cycling contributes to anticancer activity of vitamin K. NQO1 competes with enzymes that redox cycle vitamin K and catalyzes two-electron reduction of vitamin K3 to hydroquinone. This skips formation of semiquinone and ROS. Therefore, NQO1 metabolically detoxifies vitamin K3 and protects cells against oxidative stress and other adverse effects. On the contrary, NQO2 catalyzes metabolic activation of vitamin K3 leading to cytotoxicity. The role of NQO1 and NQO2 in metabolic detoxification and/or activation of vitamin K1 and K2 remains to be determined. Future studies are also required to identify the enzymes that catalyze high affinity reduction of vitamin K1 and K2 to hydroquinone for use in gamma-carboxylation reactions.