Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of Hepatitis C Virus-Infected Cells and Liver to Identify Pathways Associated With Disease Development.

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.

PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis.

We report the engineering and characterization of paraoxonase-3 knockout mice (Pon3KO). The mice were generally healthy but exhibited quantitative alterations in bile acid metabolism and a 37% increased body weight compared to the wild-type mice on a high fat diet. PON3 was enriched in the mitochondria-associated membrane fraction of hepatocytes. PON3 deficiency resulted in impaired mitochondrial respiration, increased mitochondrial superoxide levels, and increased hepatic expression of inflammatory genes. PON3 deficiency did not influence atherosclerosis development on an apolipoprotein E null hyperlipidemic background, but it did lead to a significant 60% increase in atherosclerotic lesion size in Pon3KO mice on the C57BL/6J background when fed a cholate-cholesterol diet. On the diet, the Pon3KO had significantly increased plasma intermediate-density lipoprotein/LDL cholesterol and bile acid levels. They also exhibited significantly elevated levels of hepatotoxicity markers in circulation, a 58% increase in gallstone weight, a 40% increase in hepatic cholesterol level, and increased mortality. Furthermore, Pon3KO mice exhibited decreased hepatic bile acid synthesis and decreased bile acid levels in the small intestine compared with wild-type mice. Our study suggests a role for PON3 in the metabolism of lipid and bile acid as well as protection against atherosclerosis, gallstone disease, and obesity.

Simultaneous measurement of endogenous cortisol, cortisone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in nails by use of UPLC-MS-MS.

Steroid hormone concentrations are mostly determined by using different body fluids as matrices and applying immunoassay techniques. However, usability of these approaches may be restricted for several reasons, including ethical barriers to invasive sampling. Therefore, we developed an ultra-performance LC-MS-MS method for high-throughput determination of concentrations of cortisol, cortisone, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEAS) in small quantities of human nails. The method was validated for linearity, limits of detection and quantification, recovery, intra and interassay precision, accuracy, and matrix effect. Samples from 10 adult women were analyzed to provide proof-of-principle for the method's applicability. Calibration curves were linear (r(2) > 0.999) in the ranges 10-5000 pg mg(-1) for cortisol, cortisone, and DHEAS, and 50-5000 pg mg(-1) for DHEA. Limits of quantification were 10 pg mg(-1) for cortisol, cortisone, and DHEAS, and 50 pg mg(-1) for DHEA. The sensitivity and specificity of the method were good, and there was no interference with the analytes. Mean recovery of cortisol, cortisone, DHEA, and DHEAS was 90.5%, 94.1%, 84.9%, and 95.9%, respectively, with good precision (coefficient of variation <14% for all analytes) and accuracy (relative error (%) -8.3% to 12.2% for all analytes). The median (pg mg(-1), range) hormone concentrations were 69.5 (36-158), 65 (32-133), 212 (50-1077), and 246 (115-547) for cortisol, cortisone, DHEA, and DHEAS, respectively. This method enables measurement of cortisol, cortisone, DHEA, and DHEAS in small quantities of human nails, leading to the development of applications in endocrinology and beyond.

Prenatal testosterone exposure is associated with delay of gratification and attention problems/overactive behavior in 3-year-old boys.

Sex differences in self-control become apparent during preschool years. Girls are better able to delay their gratification and show less attention problems and overactive behavior than boys. In this context, organizational effects of gonadal steroids affecting the neural circuitry underlying self-control could be responsible for these early sex differences. In the present study testosterone levels measured in amniotic fluid (via ultra performance liquid chromatography and tandem mass spectrometry) were used to examine the role of organizational sex hormones on self-control. One hundred and twenty-two 40-month-old children participated in a delay of gratification task (DoG task) and their parents reported on their attention problems and overactive behavior. Girls waited significantly longer for their preferred reward than boys, and significantly more girls than boys waited the maximum period of time, providing evidence for sex differences in delay of gratification. Boys that were rated as suffering from more attention problems and overactive behavior waited significantly shorter in the DoG task. Amniotic testosterone measures were reliable in boys only. Most importantly, boys who waited shorter in the DoG task and boys who were reported to suffer from more attention problems and overactive behavior had higher prenatal testosterone levels. These findings extend our knowledge concerning organizational effects of testosterone on the brain circuitry underlying self-control in boys, and are of relevance for understanding how sex differences in behavioral disorders are connected with a lack of self-control.

Sex specific relationships between infants' mental rotation ability and amiotic sex hormones.

Sex differences in mental rotation, robust in adults, have recently been reported for infants' looking times although the pattern of results is not completely conclusive. In this context, organizational effects of gonadal steroids affecting the neural circuitry underlying spatial cognition could be (partly) responsible for the early sex difference. In the present study testosterone and estradiol levels measured in amniotic fluid via ultra performance liquid chromatography and tandem mass spectrometry were used to examine the role of prenatal sex hormones on infants' looking times during mental rotation. N = 208 six-month-old infants participated in an expectation of violation task with 3D cube figures. Mental rotation was defined as the difference in looking times for familiar versus mirrored cube figures whereas vigilance was defined as the sum of both looking times. Sex differences were absent for mental rotation as well as for vigilance. Most importantly, however, for boys mental rotation but not vigilance was correlated with prenatal testosterone but not with estradiol. For girls mental rotation but not vigilance was correlated with prenatal estradiol but not with testosterone although it has to be noted that the testosterone values for girls suffered from a floor effect. Only 5% of the within-sex variance was due to prenatal sex hormones indicating small effects. These findings extend our knowledge concerning organizational effects of prenatal sex hormones on the brain circuitry underlying spatial cognition.

Determination of cannabinoids in whole blood by UPLC-MS-MS.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in whole blood. Samples were prepared by protein precipitation followed by solid-phase extraction. Data were acquired using positive electrospray ionization and multiple reaction monitoring. Two transitions were selected for THC (m/z 315.0 > 193.0 and m/z 315.0 > 122.7) and THC-COOH (m/z 345.0 > 299.0 and m/z 345.0 > 327.0), and one transition was chosen for 11-OH-THC (m/z 331.0 > 313.0). Deuterated analogues of each analyte were used as internal standards for quantification. Run time was 10 min. Limits of quantification (LOQ) were 0.05 ng/mL for THC, 0.1 ng/mL for 11-OH-THC, and 0.2 ng/mL for THC-COOH. Linearity was established from LOQ to 50 ng/mL for each substance (r(2) always > 0.999). Accuracy ranged from 96 to 106%, and imprecision was less than 10% for all analytes. The UPLC-MS-MS method was found to be sensitive, specific, and rapid because it requires no derivatization step. It can be an alternative to gas chromatography-mass spectrometry for the determination of cannabinoids in whole blood.

Detection of the designer benzodiazepine metizolam in urine and preliminary data on its metabolism.

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available via the Internet without control. Metizolam was ordered via the Internet and a 2 mg blue tablet was orally administered to a 54-year-old man. Urine samples were collected over 6 days in polypropylene tubes. After liquid/liquid extraction at pH 9.5, metizolam was analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) using a standard method devoted to benzodiazepines, and ions transitions, at m/z 328.9 > 275.0 and 328.9 > 300.0. Metizolam was detectable in hydrolyzed urine during the 46-h period, with concentrations always lower than 11 ng/mL. About 0.3% of the initial dose was excreted in urines as total unchanged metizolam during the first 24 h. The most relevant potential CYP- and UGT-dependent metabolites of metizolam were investigated in vitro using human liver microsome incubation and, subsequently, liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. Three mono-hydroxylated metabolites were produced including a hydroxylation compound at the 2-ethyl moiety of metizolam (M1) as quantitatively main metabolite, and a N-hydroxymetiazolam (M2). The structure of the third metabolite (M3) could not be elucidated because of a too low experimental production rate. Two authentic urine samples were analyzed using the same analytical method to search for metabolites of metizolam. M1, together with its glucuronide (M1-Glu), and M2 were observed in urine at the 8 h mark, whereas only M1 and M1-Glu were still detected in urine at 30 h post administration. Copyright © 2016 John Wiley & Sons, Ltd.

Fatal Combination with 3-Methylmethcathinone (3-MMC) and Gamma-Hydroxybutyric Acid (GHB).

We reported the case of 69-year-old man who was discovered dead at a friend's home. 3-Methylmethcathinone (3-MMC) and poppers (alkyl nitrites) were found at the scene by the police. Autopsy specimens including peripheral and cardiac blood, urine, gastric content, bile and hair were sent to our laboratory to document a possible death involving abuse of drugs. Routine toxicological analysis was performed with gas chromatography with flame ionization detection (GC-FID), high performance liquid chromatography-diode array detection (HPLC-DAD), headspace gas chromatography-mass spectrometry (HS-GC-MS), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS)-MS. After liquid-liquid extraction at alkaline pH, 3-MMC was identified with GC-MS (to allow the discrimination with 4-MMC) and quantified with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-MS with the two following transitions: m/z 178.1 > 160 and 178.1 > 144.9. Gamma-hydroxybutyric acid (GHB) was analyzed by GC-MS for fluids and GC-MS-MS for hair. Toxicological analysis in peripheral blood revealed the presence of 3-MMC (0.33 mg/L), pseudoephedrine (0.03 mg/L) and GHB (576 mg/L). These molecules have also been found in other post-mortem fluids. Furthermore, testing for "poppers" by HS-GC-MS was negative. Hair analysis, without segmentation, demonstrated the presence of 3-MMC (206.7 ng/mg), pseudoephedrine (0.16 ng/mg) and GHB (96.3 ng/mg) and suggested a repeated consumption of these substances. However, one cannot exclude contamination by sweat during the agony period. Toxicological post-mortem results suggest a fatal combination of 3-MMC and GHB. Despite his age, the decedent was known to abuse drugs.

Testing for GHB in hair by GC/MS/MS after a single exposure. Application to document sexual assault.

Gamma-hydroxybutyric acid, or GHB, is a substance naturally present within mammal species. Properties of neurotransmitter or neuromodulator are generally given to this substance. GHB is therapeutically used as an anesthetic, but can be used for criminal offenses (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. In order to document single exposure, we investigated the use of hair. Hair was collected one month after the allegated event in order to sample the corresponding period after regular growing. After rapid (2 min) decontamination with dichloromethane, the hair shaft was cut into 3-mm segments. They were overnight incubated in 0.01 N NaOH in the presence of GHB-d6, followed by neutralization and extraction in ethyl acetate under acidic conditions. GHB (precursor ion m/z 233, product ions m/z 147 and 148) was tested by GC/MS/MS (Finnigan TSQ 700) after derivatization with BSTFA + 1% TMCS. Physiological concentrations (n = 24) were in the range 0.5 to 12.0 ng/mg, with no influence due to hair color. No variation of concentrations was observed along the hair shaft in controlled subjects, except for the proximal segment, due to an incorporation through sweat. This demonstrates that endogenous levels for each single subject are constant during hair growth. A controlled human administration of 25 mg/kg to a volunteer demonstrated that a single exposure to GHB is detectable in hair after segmentation. In a case of rape under influence, a clear increase of the corresponding segment (about 2.4 ng/mg) in time was observed, in comparison with the other segments (0.6 to 0.8 ng/mg). This study demonstrates that a single exposure to GHB in a case of sexual assault can be documented by hair analysis when collected about one month after the crime.

Sexual abuse and anti-wrinkle cream: evidence from octocrylene.

We report an alleged case of sexual assault in which an anti-wrinkle cream could have been used as a lubricant. Three anorectal samples taken from the victim were given to us in an attempt to document the presence of remains of the cream involved. After examining the composition of the cream, octocrylene (OCT) was selected as the most relevant marker for this analysis. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for identification of OCT. Anorectal samples were diluted with methanol and injected onto an Acquity BEH C18 column using a gradient mode with 0.1% formic acid/acetonitrile as the mobile phase. Data were acquired using positive electrospray ionization and multiple reaction monitoring. Three transitions were selected for OCT (m/z 362.2>250.0, m/z 362.2>232.0 and m/z 362.2>204.0). The analysis of the cream seized at the offender's home confirmed the presence of OCT as an ingredient, and the analysis of extracts from the anorectal samples also allowed the formal identification of OCT. These results strongly suggest that a cosmetic containing octocrylene as an ingredient has in fact been applied to the anus of the alleged victim.

Dehydroepiandrosterone in nails of infants: a potential biomarker of intrauterine responses to maternal stress.

Easily accessible biomarkers for fetal stress biology are lacking. We here explore whether quantification of major fetal steroids, dehydroepiandrosterone (DHEA) or DHEA sulfate (DHEAS), with liquid chromatography/tandem mass spectrometry in infant nails is a tool to assess fetal stress biology in response to maternal stressful life events during pregnancy. Sufficient nail (≥ 1 mg) was available from 80 infants (93% of those providing samples). The concentration of DHEA, but not DHEAS, was increased in infants of mothers with stressful life events during pregnancy (DHEA: F1,41=6.105, P=0.018; DHEAS: F1,77=0.767, P=0.384). DHEA concentrations were not related to maternal stress before pregnancy (F1,41=0.010, P=0.922). Infant nail DHEA may be a fetal biological correlate of intrauterine exposure to maternal stress. The method promises the first non-invasive retrospective biomarker for intrauterine stress biology, opening new ways for research and clinical applications in fetal medicine, endocrinology, obstetrics, gynecology, and for understanding the developmental origins of health and disease.