Rates of substance and polysubstance use through universal maternal testing at the time of delivery.

OBJECTIVE: To report substance and polysubstance use at the time of delivery. STUDY DESIGN: A cross-sectional study was performed on mothers consented for universal drug testing (99%) during hospital admission at six delivery hospitals in Cincinnati, Ohio. Mass spectrometry urinalysis detected positivity rates of 46 substances. Rates of positive drug tests for individual and common co-occurring substances measured were reported. RESULTS: 2531 maternal samples were tested (88%) and 33% contained cotinine, 11.3% THC, 7.2% opioids, 3.8% cocaine, and 1.9% methamphetamines. Polysubstance use prevalence was as high as 15%. Among mothers testing positive for methadone or buprenorphine, 93% also tested positive for cotinine and 39% tested positive for a third substance in addition to cotinine. CONCLUSIONS: Substance use at delivery is more prevalent than previously reported. Many mothers testing positive for opioids also test positive for other substances, which may increase overdose risk and exacerbate neonatal opioid withdrawal syndrome (NOWS).

Regional comparison of self-reported late pregnancy cigarette smoking to mass spectrometry analysis.

OBJECTIVE: To report a more accurate prevalence estimate of late pregnancy nicotine exposures. STUDY DESIGN: A cross-sectional study during a 2-month period in 2019. Participants were women delivering in any of the six county maternity hospitals who consented to universal drug testing at the time of delivery as part of routine hospital admission. RESULTS: Of 2531 tested samples, 18.7% tested positive for high levels of cotinine indicating primary smoking or other primary use of tobacco products. Together, 33.0% of the study population tested positive for nicotine exposure during late pregnancy compared to vital records which reported 8.2% cigarette smoking during the third trimester of pregnancy and 10.5% cigarette smoking at any time during pregnancy through maternal self-report. CONCLUSION: Captured vital birth smoking measures vastly underreport actual primary exposures to nicotine products. Vital birth data also fail to capture secondhand exposures which constitute a significant proportion of the population.

Stable-Isotope Dilution HPLC-Electrospray Ionization Tandem Mass Spectrometry Method for Quantifying Hydroxyurea in Dried Blood Samples.

BACKGROUND: Sickle cell anemia (SCA) is a life-threatening blood disorder characterized by the presence of sickle-shaped erythrocytes. Hydroxyurea is currently the only US Food and Drug Administration-approved treatment and there is a need for a convenient method to monitor compliance and hydroxyurea concentrations, especially in pediatric SCA patients. METHODS: We describe a novel approach to the determination of hydroxyurea concentrations in dried whole blood collected on DMPK-C cards or volumetric absorptive microsampling (VAMS) devices. Hydroxyurea was quantified by electrospray ionization LC-MS/MS using [13C15N2]hydroxyurea as the internal standard. Calibrators were prepared in whole blood applied to DMPK-C cards or VAMS devices. RESULTS: Calibration curves for blood hydroxyurea measured from DMPK-C cards and VAMS devices were linear over the range 0.5-60 µg/mL. Interassay and intraassay CVs were <15% for blood collected by both methods, and the limit of detection was 5 ng/mL. Whole blood hydroxyurea was stable for up to 60 days on DMPK-C cards and VAMS devices when frozen at -20 °C or -80 °C. Whole blood hydroxyurea concentrations in samples collected on DMPK-C cards or VAMS devices from SCA patients were in close agreement. CONCLUSIONS: This tandem mass spectrometry method permits measurement of hydroxyurea concentrations in small volumes of dried blood applied to either DMPK-C cards or VAMS devices with comparable performance. This method for measuring hydroxyurea from dried blood permits the evaluation of therapeutic drug monitoring, individual pharmacokinetics, and medication adherence using heel/finger-prick samples from pediatric patients with SCA treated with hydroxyurea.

Suppression of pulmonary CYP2A13 expression by carcinogen-induced lung tumorigenesis in a CYP2A13-humanized mouse model.

CYP2A13 is a human cytochrome P450 (P450) enzyme important in the bioactivation of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13 expression levels vary dramatically among lung biopsy samples from patients, presumably owing in part to a suppression of CYP2A13 expression by disease-associated inflammation. Here, we determined whether CYP2A13 expression in the lungs of CYP2A13-humanized mice is suppressed by the presence of lung tumors. Tissues from an NNK lung tumor bioassay were examined. CYP2A13-humanized mice (95-100%) had multiple lung tumors at 16 weeks after NNK (30 or 50 mg/kg) treatment; whereas only ∼9% of saline-treated CYP2A13-humanized mice had lung tumor (∼1/lung). Mice with lung tumors, from the NNK-treated groups, were used for dissecting adjacent tumor-free lung tissues; whereas mice without visible lung tumors, from the saline-treated group, were used as controls. Compared with the controls, the levels of CYP2A13 protein and mRNA were both reduced significantly (by ≥50%) in the NNK-treated groups. The levels of mouse CYP2B10 and CYP2F2 mRNAs were also significantly lower in the dissected normal lung tissues from tumor-bearing mice than in lungs from the control mice. Pulmonary tissue levels of three proinflammatory cytokines, tumor necrosis factor alpha, interferon gamma, and interleukin-6, were significantly higher in the tumor-bearing mice than in the controls, indicating occurrence of low-grade lung inflammation at the time of necropsy. Taken together, these findings support the hypothesis that CYP2A13 levels in human lungs can be suppressed by disease-associated inflammation in tissue donors, a scenario causing underestimation of CYP2A13 levels in healthy lungs.

Identification of cytochrome P450 enzymes critical for lung tumorigenesis by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): insights from a novel Cyp2abfgs-null mouse.

Cytochrome P450 (P450) enzymes encoded by the mouse Cyp2abfgs gene cluster are preferentially expressed in the respiratory tract. Previous studies have demonstrated that pulmonary P450-mediated bioactivation is necessary for lung tumorigenesis induced by the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and that CYP2A5 mediates a noteworthy fraction, but not all, of NNK bioactivation in the lung. The aim of this study was to determine whether other P450s encoded by the Cyp2abfgs gene cluster also play significant roles in NNK lung tumorigenesis. A novel Cyp2abfgs-null mouse was generated, in which all Cyp2a, 2b, 2g, 2f and 2s genes are deleted. The Cyp2abfgs-null mouse was viable, fertile and without discernible physiological abnormalities or compensatory increases in the expression of other P450s. NNK bioactivation in vitro and NNK-induced DNA adduction and lung tumorigenesis in vivo were determined for wild-type (WT) and Cyp2abfgs-null mice; the results were compared with previous findings from Cyp2a5-null mice. The Cyp2abfgs-null mice exhibited significantly lower rates of NNK bioactivation in lung and liver microsomes, compared with either WT or Cyp2a5-null mice. The levels of lung O(6)-methyl guanine DNA adduct were also substantially reduced in Cyp2abfgs-null mice, compared with either WT or Cyp2a5-null mice. Moreover, the Cyp2abfgs-null mice were largely resistant to NNK-induced lung tumorigenesis at both low (50mg/kg) and high (200mg/kg) NNK doses, in contrast to the WT or Cyp2a5-null mice. These results indicate for the first time that, collectively, the CYP2A, 2B, 2F, 2G, and 2S enzymes are indispensable for NNK-induced lung tumorigenesis.

Role of hepatic and intestinal p450 enzymes in the metabolic activation of the colon carcinogen azoxymethane in mice.

P450-mediated bioactivation of azoxymethane (AOM), a colon carcinogen, leads to the formation of DNA adducts, of which O(6)-methylguanine (O(6)-mG) is the most mutagenic and contributes to colon tumorigenesis. To determine whether P450 enzymes of the liver and intestine both contribute to AOM bioactivation in vivo, we compared tissue levels of AOM-induced DNA adducts, microsomal AOM metabolic activities, and incidences of colonic aberrant crypt foci (ACF) among wild-type (WT), liver-specific P450 reductase (Cpr)-null (LCN), and intestinal epithelium-specific Cpr-null (IECN) mice. At 6 h following AOM treatment (at 14 mg/kg, s.c.), O(6)-mG and N(7)-mG levels were highest in the liver, followed by the colon, and then small intestine in WT mice. As expected, hepatic adduct levels were significantly lower (by >60%) in LCN mice but unchanged in IECN mice, whereas small-intestinal adduct levels were unchanged or increased in LCN mice but lower (by >50%) in IECN mice compared to that in WT mice. However, colonic adduct levels were unchanged in IECN mice compared to that in WT mice and increased in LCN mice (by 1.5-2.9-fold). The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed by rates of formation of formaldehyde and N(7)-mG in vitro. Furthermore, the incidence of ACF, a lesion preceding colon cancer, was similar in the three mouse strains. Thus, AOM-induced colonic DNA damage and ACF formation is not solely dependent on either hepatic or intestinal microsomal P450 enzymes. P450 enzymes in both the liver and intestine likely contribute to AOM-induced colon carcinogenesis.

Role of CYP2A13 in the bioactivation and lung tumorigenicity of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: in vivo studies using a CYP2A13-humanized mouse model.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is abundant in tobacco smoke, is a potent lung procarcinogen. The present study was aimed to prove that transgenic expression of human cytochrome P450 2A13 (CYP2A13), known to be selectively expressed in the respiratory tract and be the most efficient enzyme for NNK bioactivation in vitro, will enhance NNK bioactivation and NNK-induced tumorigenesis in the mouse lung. Kinetic parameters of NNK bioactivation in vitro and incidence of NNK-induced lung tumors in vivo were determined for wild-type, Cyp2a5-null and CYP2A13-humanized (CYP2A13-transgenic/Cyp2a5-null) mice. As expected, in both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in Michaelis constant (K m) values for the formation of 4-oxo-4-(3-pyridyl)-butanal, representing the reactive intermediate that can lead to the formation of O(6)-methylguanine (O(6)-mG) DNA adducts; however, the gain of CYP2A13 at a fraction of the level of mouse lung CYP2A5 led to recovery of the activity in the lung, but not in the liver. The levels of O(6)-mG, the DNA adduct highly correlated with lung tumorigenesis, were significantly higher in the lungs of CYP2A13-humanized mice than in Cyp2a5-null mice. Moreover, incidences of lung tumorigenesis were significantly greater in CYP2A13-humanized mice than in Cyp2a5-null mice, and the magnitude of the differences in incidence was greater at low (30mg/kg) than at high (200mg/kg) NNK doses. These results indicate that CYP2A13 is a low K m enzyme in catalyzing NNK bioactivation in vivo and support the notion that genetic polymorphisms of CYP2A13 can influence the risks of tobacco-induced lung tumorigenesis in humans.

Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).

BACKGROUND: Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response. OBJECTIVES: To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M. METHODS: The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (µmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17ß-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level. RESULTS: The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT. CONCLUSION: SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Hepatobiliary disposition of 3alpha,6alpha,7alpha,12alpha-tetrahydroxy-cholanoyl taurine: a substrate for multiple canalicular transporters.

Tetrahydroxy bile acids become major biliary bile acids in Bsep(-/-) mice and Fxr(-/-) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(-/-) mice in the presence or absence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(-/-) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(-/-), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05-0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (K(m) = 0.13 mM) than for mMdr1a (K(m) = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(-/-) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.

Human multidrug resistance protein 2 transports the therapeutic bile salt tauroursodeoxycholate.

The multidrug resistance protein 2 (MRP2/ABCC2) mediates the biliary excretion of glucuronide and glutathione conjugates of endogenous and exogenous compounds. We examined the activation of human MRP2-mediated ATP-dependent transport by the choleretic bile salt ursodeoxycholic acid (UDC) and its taurine and glycine amidates in Sf9 cell membranes expressing MRP2 using beta-estradiol 17-(beta-D-glucuronide) (E(2)17G) and beta-estradiol 3-(beta-D-glucuronide) (E(2)3G) as substrates. MRP2 transported E(2)3G via classic Michaelis-Menten kinetics (K(m) = 122 microM; V(max) = 3.0 nmol/mg/min), whereas E(2)17G transport showed positive cooperativity (Hill slope, 2.15; K(m) = 75 microM; V(max) = 3.8 nmol/mg/min). UDC, tauroursodeoxycholate, and glycoursodeoxycholate (80-100 microM) maximally stimulated E(2)3G transport 9-, 7.9-, and 3.6-fold, respectively, whereas higher concentrations (1-2 mM) inhibited transport. At low (0.3 microM) concentrations, tauroursodeoxycholate was transported only in the presence of E(2)17G or E(2)3G, but not other MRP2 substrates such as methotrexate, leukotriene C(4), or S-methylglutathione. Kinetic analysis of higher concentrations of tauroursodeoxycholate transport by MRP2 showed positive cooperativity (Hill slope, 1.84; K(m) = 127 microM; V(max) = 779 pmol/mg/min). Taurocholate (2-100 microM) was not detectably transported by MRP2 either alone or in the presence of E(2)17G but was transported in the presence of E(2)3G. Thus, UDC, tauroursodeoxycholate, and glycoursodeoxycholate activated MRP2 transport. Tauroursodeoxycholate was transported by MRP2 and demonstrated positive cooperativity, identifying it as the second MRP2 substrate able to stimulate its own transport. The data suggest MRP2 binding sites that can require specific complementarities between substrates and modulators of MRP2-mediated transport.