Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

Background There is a strong relationship between circadian rhythms and ethanol responses. Ethanol consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Spanagel et al., 2005b). The Drosophila central circadian pacemaker is composed of proteins encoded by the per, tim, cyc, and Clk genes. Using Drosophila mutant analysis we asked whether these central components of the circadian clock make the equivalent contribution towards ethanol tolerance and whether rhythmicity itself is necessary for tolerance. Methods We tested flies carrying mutations in core clock genes for the capacity to acquire ethanol tolerance. Tolerance was assayed by comparing the sedation curves of populations during their first and second sedation. Animals that had acquired tolerance sedated more slowly. Movement was also monitored as the flies breathe the ethanol vapor to determine if other facets of the ethanol response were affected by the mutations. Gas chromatography was used to measure internal ethanol concentration. Constant light was used to non-genetically destabilize the PER and TIM proteins. Results A group of circadian mutations, all of which eliminate circadian rhythms, do not disrupt tolerance identically. Mutations in per, tim, and cyc completely block tolerance. However, a mutation in Clk does not interfere with tolerance. Constant light also disrupts the capacity to acquire tolerance. These lines did not differ in ethanol absorption. Conclusions Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid ethanol tolerance. However, the role of circadian genes in ethanol tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire ethanol tolerance by some circadian mutations is not merely a downstream effect of a nonfunctional circadian clock, instead these circadian genes play an independent role in ethanol tolerance.

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.

Background: N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH.Methods: Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects.Results: We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant.Conclusions: Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila.

Homeostatic neural adaptations to alcohol underlie the production of alcohol tolerance and the associated symptoms of withdrawal. These adaptations have been shown to persist for relatively long periods of time and are believed to be of central importance in promoting the addictive state. In Drosophila, a single exposure to alcohol results in long-lasting alcohol tolerance and symptoms of withdrawal following alcohol clearance. These persistent adaptations involve mechanisms such as long-lasting changes in gene expression and perhaps epigenetic restructuring of chromosomal regions. Histone modifications have emerged as important modulators of gene expression and are thought to orchestrate and maintain the expression of multi-gene networks. Previously genes that contribute to tolerance were identified as those that show alcohol-induced changes in histone H4 acetylation following a single alcohol exposure. However, the molecular mediator of the acetylation process that orchestrates their expression remains unknown. Here we show that the Drosophila ortholog of mammalian CBP, nejire, is the histone acetyltransferase involved in regulatory changes producing tolerance—alcohol induces nejire expression, nejire mutations suppress tolerance, and transgenic nejire induction mimics tolerance in alcohol-naive animals. Moreover, we observed that a loss-of-function mutation in the alcohol tolerance gene slo epistatically suppresses the effects of CBP induction on alcohol resistance, linking nejire to a well-established alcohol tolerance gene network. We propose that CBP is a central regulator of the network of genes underlying an alcohol adaptation.

One of the most widely studied ultimate carcinogens, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I), is fairly stable in organic solvents but hydrolyses rapidly in the presence of water. Routine use of BPDE-I stock solutions involves multiple cycles of warming to room temperature and return to freezer temperature and results in some decomposition of the stock with time, presumably due to uptake of water from the atmosphere. We present a simple spectrophotometric assay which allows an investigator to quickly and safely estimate the extent of such decomposition to an accuracy of +/- 6%. The assay is based on the rapid reaction of BPDE-I with beta-mercaptoethanol in alkaline solution resulting in an adduct which exhibits a significant red-shift in the absorption spectrum.

The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.

The binding of chemical carcinogens to nuclear macromolecules, especially to DNA, is thought to be central to the initiation of carcinogenesis. Previous studies of the interactions of one such ultimate carcinogen, benzo[a]pyrene diol epoxide (BPDE-I) with nuclei, chromatin and purified DNA, demonstrated that although some BPDE-I-DNA interactions were altered in chromatin, covalent binding to chromatin DNA at saturating chromatin concentrations was quantitatively the same as binding to purified DNA. We have now extended these studies to include the basic subunit of chromatin, the nucleosomal core particle. Association constants for BPDE-I and a nonreactive analogue were determined by absorbance and fluorescence spectroscopy using either core particles or purified DNA and were found to be lower, by a factor of 30, for core particles. One of the major pathways of interaction of BPDE-I with DNA is the catalysis of BPDE-I hydrolysis by the exocyclic amino group of deoxyguanosine in native DNA. This detoxification reaction is inhibited about 30-fold in core particles compared with DNA, consistent with the hypothesis that intercalation is important in this catalytic reaction. In contrast to these findings, at DNA concentrations that allow maximal binding, similar amounts of BPDE-I are bound covalently to either free DNA or the DNA contained in core particles. This finding suggests that the interaction of DNA with histones to form the subunit structure of chromatin does not significantly protect DNA from damage by this ultimate carcinogen. The pattern of DNA adducts formed with core particle DNA shows a subtle shift toward the pattern seen with denatured DNA.