A case control study investigating the methylation levels of GHRL and GHSR genes in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is a relapsing disease described as excessive use of alcohol. Evidence of the role of DNA methylation in addiction is accumulating. Ghrelin is an important peptide known as appetite hormone and its role in addictive behavior has been identified. Here we aimed to determine the methylation levels of two crucial genes (GHRL and GHSR) in ghrelin signaling and further investigate the association between methylation ratios and plasma ghrelin levels. METHODS: Individuals diagnosed with (n = 71) and without (n = 82) AUD were recruited in this study. DNA methylation levels were measured through methylation-sensitive high-resolution melting (MS-HRM). Acylated ghrelin levels were detected by ELISA. The GHRL rs696217 polymorphism was analyzed by the standard PCR-RFLP method. RESULTS: GHRL was significantly hypermethylated (P < 0.0022) in AUD between 25 and 50% methylation than in control subjects but no significant changes of GHSR methylation were observed. Moreover, GHRL showed significant positive correlation of methylation ratio between 25 and 50% with age. A significant positive correlation between GHSR methylation and ghrelin levels in the AUD group was determined (P = 0.037). The level of GHRL methylation and the ghrelin levels showed a significant association in the control subjects (P = 0.042). CONCLUSION: GHSR and GHRL methylation levels did not change significantly between control and AUD groups. However, GHRL and GHSR methylations seemed to have associations with plasma ghrelin levels in two groups. This is the first study investigating the DNA methylation of GHRL and GHSR genes in AUD.

Effect of MAOA rs1465108 polymorphism on susceptibility to substance/alcohol use disorder: a novel PCR-RFLP assay for the detection of MAOA rs1465108.

BACKGROUND: The health and social consequences of substance/alcohol use disorders are harmful. Most of the individuals cannot stop using them due to more likely their genetic background. The current study aimed both to develop a novel PCR-RFLP method for genotyping of MAOA rs1465108 and to analyze the effect of MAOA rs1465108 on the risk of alcohol (AUD), opioid (OUD) or methamphetamine (MUD) use disorders and on the depressive and anxiety symptoms in a Turkish population. METHODS AND RESULTS: A total of 353 individual with AUD (n = 154), OUD (n = 160) or MUD (n = 39) and 109 healthy subjects were included. The intensity of anxiety and depressive symptoms and craving and opioid withdrawal were measured by appropriate scales. Logistic regression analysis revealed no association between MAOA rs1465108 polymorphism and substance/alcohol use disorder (p > 0.05). Healthy subjects (3.0) had significantly lower levels of depressive symptoms than individuals with OUD (27.0), AUD (21.0) and MUD (25.5) groups. The severity of depressive symptoms was significantly higher in OUD as compared to AUD. There was a statistically significant difference between individuals with AUD, OUD and MUD in view of the average ages of first use (17, 19 and 20 years, respectively) (p < 0.05). CONCLUSIONS: The results presented here do not support the hypothesis that MAOA rs1465108 is associated with substance/alcohol use disorders. The intensity of depressive symptoms could be changed according to the abused substance type. A novel PCR-RFLP was developed for genotyping of MAOA rs1465108 polymorphism, which could be a better option for laboratories without high technology equipment.

Effect of PDYN and OPRK1 polymorphisms on the risk of alcohol use disorder and the intensity of depressive symptoms.

AIMS: The dynorphin (DYN)/Kappa Opioid Receptor (KOR) system has been suggested to be involved in both negative affective states and the action of alcohol. The present study was undertaken to explore whether the DYN/KOR system genes, PDYN and OPRK1, influence on individual differences in the intensity of depressive symptoms at admission as well as the risk of alcohol use disorder (AUD) risk in a sample of 101 individuals with AUD and 100 controls. METHODS: PDYN (rs2281285, rs2225749 and rs910080) and OPRK1 (rs6473797, rs963549 and rs997917) polymorphisms were analyzed by PCR-RFLP. The intensity of depressive and anxiety symptoms and craving were measured by the Beck Depression Inventory-II (BDI-II), Beck Anxiety Inventory (BAI), and Penn Alcohol Craving Scale, respectively. RESULTS: A significant association between the risk of AUD and OPRK1 rs6473797 (P < 0.05) at the gene level. OPRK1 rs6473797 CC genotype was found to lead to a 3.11 times greater alcohol dependence risk. In addition, the BDI-II score of the OPRK1 rs963549 CC genotype was found to be significantly lower (20.9 ± 11.2, min: 1.0, max: 48.0) than that of the CT + TT genotypes (27.04 ± 12.7, min: 0.0, max: 49.0) (t: -2.332, P = 0.022). None of the PDYN polymorphisms were associated with BDI-II score. CONCLUSION: Variations in the KOR are associated with the risk of AUD and the intensity of depressive symptoms at admission at the gene level in Turkish males. On the other hand, PDYN gene seemed not to be associated with AUD, depression, anxiety, and craving.

OPRM1 rs2075572 has potential to affect plasma buprenorphine level in opioid users, but not OPRM1 rs562859.

OPRM1 gene encoding mu-opioid receptor (MOR) is the primary candidate gene for buprenorphine (BUP) pharmacogenetics. OPRM1 undergoes alternative splicing leading to multiple MOR subtypes. Thus, in the current study 2 SNPs (rs1799972 and rs562859) were selected due to evidence for their contribution to alternative splicing of OPRM1. The effects of 2 SNPs of OPRM1 gene on plasma buprenorphine and norbuprenorphine levels in a sample of 233 OUD patients receiving BUP/naloxone were examined. Polymorphisms were analyzed by PCR and RFLP. BUP and norbuprenorphine concentrations in plasma were measured by LC-MS/MS. OPRM1 rs2075572 GC + CC (0.12 ng/ml) had significantly higher plasma BUP level compared to GG (0.084 ng/ml) (p = 0.043). Although there was not a statistically significant difference between OPRM1 rs562859 genotypes (p = 0.46), patients with OPRM1 rs562859 CT + TT had higher plasma BUP and BUP-related values as compared to those with CC. In conclusion, the effect of OPRM1 rs2075572 on BUP levels in opioid users' plasma was shown in a Caucasian population for the first time. On the other hand, OPRM1 rs562859 seems not to influence the BUP pharmacology.

Association of OPRK1 rs963549 and rs997917 polymorphisms with opioid use disorder and related phenotypes.

Aim: To evaluate the association between OPRK1 rs963549 and rs997917 and opioid use disorder (OUD) and related phenotypes. Methods: A sample of 208 individuals with (n = 100) and without (n = 108) OUD were enrolled. OPRK1 rs963549 and rs997917 were analyzed by PCR-RFLP. Craving, opioid withdrawal and the intensity of depressive and anxiety symptoms were measured by the appropriate scales. Results: OPRK1 rs963549 variation showed a trend of association with decreased opioid withdrawal. No significant associations were found between OPRK1 rs963549 and rs997917 polymorphisms and craving, depression or anxiety symptoms. Neither single OPRK1 SNPs nor OPRK1 haplotypes were associated with OUD. Conclusion: Our results could be useful for treatment failures of individuals who experience greater opioid withdrawal due to their OPRK1 rs963549 genotypes.

OPRD1 rs569356 polymorphism has an effect on plasma norbuprenorphine levels and dose/kg-normalized norbuprenorphine values in individuals with opioid use disorder.

This study aimed to determine the effects of nine OPRM1, OPRD1 and OPRK1 polymorphisms on plasma BUP and norbuprenorphine (norBUP) concentrations and various treatment responses in a sample of 122 patients receiving BUP/naloxone. Plasma concentrations of BUP and norBUP were detected by LC-MS/MS. PCR-RFLP method was used to genotype polymorphisms. OPRD1 rs569356 GG had significantly lower plasma norBUP concentration (p = 0.018), dose- (p = 0.049) and dose/kg-normalized norBUP values (p = 0.036) compared with AA. Craving and withdrawal symptoms were significantly higher in OPRD1 rs569356 AG+GG relative to AA. There was a statistically significant difference between the OPRD1 rs678849 genotypes in the intensity of anxiety (13.5 for CT+TT and 7.5 for TT). OPRM1 rs648893 TT (18.8 ± 10.8) was significantly different to CC+CT (14.82 ± 11.3; p = 0.049) in view of the intensity of depression. This current study provides the first data on a prominent effect of the OPRD1 rs569356 variation on BUP pharmacology due to its metabolite norBUP.

Are BPA-free plastics safe for aquatic life? - Fluorene-9-bisphenol induced thyroid-disrupting effects and histopathological alterations in adult zebrafish (Danio rerio).

Fluorene-9-bisphenol (BPFL) is used as an alternative compound for bisphenol A, an endocrine disruptor compound which is present in various materials including plastic bottles and packaging. Although it is used extensively in products that are labelled BPA-free, its effect on wildlife and humans have not been fully studied. Therefore, this study aimed to investigate the effects of BPFL in adult zebrafish. In the preliminary experiments of the study, the median lethal concentration value (LC50) of BPFL was 0.25 mg/L (95 % confidence interval 0.15-0.41) for 96 h. Following exposure to three different sublethal concentrations of BPFL after 96 h and 15 days, T4 hormone levels, expression levels of genes involved in thyroid metabolism and histopathological alterations were assessed. T4 hormone levels were found to be significantly higher in females at the lowest BPFL concentration following 96 h exposure (P < 0.05). Expression levels of trh, tshba and trhrb genes were upregulated following 96 h exposure at 0.025 mg/L concentration and crh was upregulated following 15 days exposure at 0.025 mg/L concentration in female zebrafish (P < 0.05). The most prominent histopathological findings in zebrafish exposed to 0.025 and 0.125 mg/L of BPFL were observed in the gill, liver, kidney and testis tissues. The gill tissues showed some hyperemia, lamellar fusion, hyperplasia, epithelial lifting, and telangiectasis, while passive hyperemia, hydropic degeneration, and necrosis were observed in the liver tissues. The BPFL is highly toxic to zebrafish even in sublethal concentrations according to the molecular and histopathological responses.

Sublingual buprenorphine/naloxone treatment is not affected by OPRM1 A118G and BDNF Va66Met polymorphisms, but alters the plasma beta-endorphin and BDNF levels in individuals with opioid use disorder.

The study aimed to examine the genetic contribution to buprenorphine (BUP) treatment in individuals with opioid use disorder (OUD), with a specific focus on BDNF and OPRM1 genes. A total of 113 controls and 111 OUD patients receiving sublingual BUP/naloxone were enrolled. OPRM1 A118G and BDNF Val66Met polymorphisms were investigated by PCR-FRLP. Plasma BDNF and beta-endorphin levels were assessed by ELISA kits in both groups. Blood BUP levels were measured by LC-MS/MS and normalized with daily BUP dose (BUP/D). OPRM1 A118G and BDNF Val66Met polymorphisms didn't have an effect on plasma beta-endorphin and BDNF levels in OUD patients, respectively. Interestingly, OUD patients had significantly higher plasma BDNF and lower beta-endorphin levels compared to the controls (p < 0.001). A negative and significant correlation between plasma BUP/D and BDNF levels was found. Age onset of first use was associated with OPRM1 A118G polymorphism. The findings indicated that sublingual BUP/naloxone may increase plasma BDNF levels, but may decrease beta-endorphin levels in individuals with OUD. Plasma BDNF level seemed to be decreased in a BUP/D concentration-dependent manner.

Effects of UGT2B7 rs7662029 and rs7439366 polymorphisms on sublingual buprenorphine metabolism in heroin addicts: An improved PCR-RFLP assay for the detection of rs7662029 polymorphism.

This study aimed to determine the effects of UGT2B7 rs7662029 and rs7439366 polymorphisms on plasma buprenorphine (BUP) concentration and different treatment responses in a sample of 109 patients with opioid use disorder (OUD) treated with sublingual BUP/naloxone. Polymorphisms were analysed by PCR-RFLP. Plasma concentrations of BUP and its metabolite norbuprenorphine were detected by LC-MS/MS. Craving, withdrawal, depression and anxiety were measured by appropriate scales. OUD patients with rs7439366 CC or rs7662029 GG genotypes had significantly lower dose-normalized (BUP/D) and dose/kg-normalized BUP (BUP/D.kg-1) levels than those who were CT or AA carriers. Significant associations between UGT2B7 rs7662029 and increased craving (p = 0.037) and withdrawal symptoms (p = 0.029) were detected. Our findings were pointing to an important role of UGT2B7 in the metabolism of sublingual BUP/naloxone in the heroin addicts for the first time. A novel PCR-RFLP assay was developed for the determination of UGT2B7 rs7662029 polymorphism, based on utilizing novel restriction enzyme.

Association of PDYN 68-bp VNTR polymorphism with sublingual buprenorphine/naloxone treatment and with opioid or alcohol use disorder: Effect on craving, depression, anxiety and age onset of first use.

In this case-control study (423 Turkish subjects), the functional pro-dynorphin (PDYN) 68-bp VNTR polymorphism was genotyped in opioid users receiving sublingual buprenorphine/naloxone treatment (SBNT; n = 129, 119 males and 10 females), in opioid users (OUD; n = 99, 90 males and 9 females), in alcohol users (AUD; n = 75, 75 males) and in controls (n = 120, 109 males and 11 females) to determine the effect of this polymorphism on different treatment responses, heroin or alcohol dependence as well as age onset of first use. The PDYN 68-bp alleles were determined based on the number of repeats and genotypes were classified as "short/short (SS)", "short-long (SL)" and "long-long (LL)". The intensity of craving, withdrawal, depression and anxiety were measured by the Substance Craving Scale (SCS), the Clinical Opiate Withdrawal Scale (COWS), the Beck Depression Inventory-II (BDI-II) and Beck Anxiety Inventory (BAI), respectively. Healthy controls (5.5 ± 5.8) had significantly lower levels of depressive symptoms compared to OUD (25.4 ± 13.5), AUD (22.5 ± 11.3) and SBNT (19.29 ± 12.2) groups. In OUD group, the LL genotype was associated with decreased intensity of anxiety and depressive symptoms than the SS+SL genotype. The BDI-II scores for PDYN VNTR genotypes within the 4 groups were analysed by two-way ANOVA and statistical differences were found for the groups. SBNT group had significantly lower COWS score than OUD group (1.00 versus 3.00). There were statistically significant differences in the median BAI (11 versus 24) and BDI-II scores (17.5 versus 25) between OUD and SBNT groups, supporting the antidepressant and anxiolytic effects of SBNT in persons with OUD.

Multiplex Detection of Aspergillus fumigatus Mycoviruses.

Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version of PCR that enables amplification of different targets simultaneously. This technique has been widely used for detection and differentiation of viruses especially plant viruses such as those which infect tobacco, potato and garlic. For rapid detection, multiplex RT-PCR was developed to screen new isolates for the presence of A. fumigatus mycoviruses. Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus (AfuPV-1), and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) dsRNAs were amplified in separate reactions using a mixture of multiplex primer pairs. It was demonstrated that in the presence of a single infection, primer pair mixtures only amplify the corresponding single virus infection. Mixed infections using dual or triple combinations of dsRNA viruses were also amplified simultaneously using multiplex RT-PCR. Up until now, methods for the rapid detection of Aspergillus mycoviruses have been restricted to small scale dsRNA extraction approaches which are laborious and for large numbers of samples not as sensitive as RT-PCR. The multiplex RT-PCR assay developed here will be useful for studies on determining the incidence of A. fumigatus mycoviruses. This is the first report on multiplex detection of A. fumigatus mycoviruses.