Maternal caffeine intake during pregnancy and the risk of delivering a small for gestational age baby: Kuopio Birth Cohort.

PURPOSE: High caffeine intake during pregnancy is associated with restricted fetal growth. We aimed to evaluate the association between maternal caffeine intake during early and late pregnancy and the risk of delivering a small for gestational age (SGA) baby. METHODS: Kuopio Birth Cohort (KuBiCo) is a prospective cohort study including women whose pregnancies and deliveries were treated at the prenatal clinics in outpatient healthcare centers and in Kuopio University Hospital, Finland. Maternal diet and caffeine intake during the first (n = 2007) and third (n = 4362) trimester of pregnancy were assessed using a 160-item food frequency questionnaire (2013-2022). SGA was defined as birth weight corrected for gestational age below - 2 standard deviations from the mean, according to the sex-specific Finnish fetal growth curves. RESULTS: Altogether in 32 and 38% (1st and 3rd trimester) of all women and in 44 and 52% of coffee drinkers, caffeine intake exceeded the recommendation for caffeine intake ( ≤ 200 mg/day) during pregnancy. The women with moderate (51-200 mg/day) (aOR 1.87; 95% CI: 1.16-3.02) and high (> 200 mg/day) (aOR 1.51; 95% CI: 1.08-2.10) caffeine intake during the first trimester were in the highest risk of having an SGA newborn. Caffeine intake in the third trimester of pregnancy was not associated with SGA. CONCLUSIONS: Moderate and high caffeine intake during early pregnancy is associated with SGA. As the results suggest that even moderate caffeine intake during the first trimester may increase the risk of SGA, the intake within recommendation limits does not necessarily appear to be safe for pregnant women and their newborns.

Placental ion channels: potential target of chemical exposure.

The placenta is an important organ for the exchange of substances between the fetus and the mother, hormone secretion, and fetoplacental immunological defense. Placenta has an organ-specific distribution of ion channels and trophoblasts, and placental vessels express a large number of ion channels. Several placental housekeeping activities and pregnancy complications are at least partly controlled by ion channels, which are playing an important role in regulating hormone secretion, trophoblastic homeostasis, ion transport, and vasomotor activity. The function of several placental ion channels (Na, Ca, and Cl ion channels, cation channel, nicotinic acetylcholine receptors, and aquaporin-1) is known to be influenced by chemical exposure, i.e., their responses to different chemicals have been tested and confirmed in experimental models. Here, we review the possibility that placental ion channels are targets of toxicological concern in terms of placental function, fetal growth, and development.

Caffeine content in newborn hair correlates with maternal dietary intake.

PURPOSE: High-maternal caffeine intake during pregnancy may be harmful for perinatal outcomes and future child health, but the level of fetal cumulative exposure has been difficult to measure thus far. Here, we present maternal dietary caffeine intake during the last trimester and its correlation to caffeine content in newborn hair after birth. METHODS: Maternal third trimester diets and dietary caffeine intake were prospectively collected in Kuopio Birth Cohort (KuBiCo) using a 160-item food frequency questionnaire (n = 2840). Newborn hair was collected within 48 h after birth and analyzed by high-resolution mass spectrometry (HRMS) for caffeine (n = 316). Correlation between dietary caffeine intake and neonatal hair caffeine content was evaluated from 203 mother-child pairs. RESULTS: Mean dietary caffeine intake was 167 mg/days (95% CI 162-172  mg/days), of which coffee comprised 81%. Caffeine in the maternal diet and caffeine content in newborn hair correlated significantly (r = 0.50; p < 0.001). Older, multiparous, overweight women, and smokers had the highest caffeine levels in the maternal diet, as well as in their newborn babies' hair. CONCLUSION: Caffeine exposure, estimated from newborn hair samples, reflects maternal third trimester dietary caffeine intake and introduces a new method to assess fetal cumulative caffeine exposure. Further studies to evaluate the effects of caffeine exposure on both perinatal and postnatal outcomes are warranted, since over 40% of pregnant women consume caffeine more than the current suggested recommendations (European Food Safety Association, EFSA recommendations).

In Vitro Human Metabolism and Inhibition Potency of Verbascoside for CYP Enzymes.

Verbascoside is found in many medicinal plant families such as Verbenaceae. Important biological activities have been ascribed to verbascoside. Investigated in this study is the potential of verbascoside as an adjuvant during tuberculosis treatment. The present study reports on the in vitro metabolism in human hepatic microsomes and cytosol incubations as well as the presence and quantity of verbascoside within Lippia scaberrima. Additionally, studied are the inhibitory properties on human hepatic CYP enzymes together with antioxidant and cytotoxic properties. The results yielded no metabolites in the hydrolysis or cytochrome P450 (CYP) oxidation incubations. However, five different methylated conjugates of verbascoside could be found in S-adenosylmethionine incubation, three different sulphate conjugates with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) incubation with human liver samples, and very low levels of glucuronide metabolites after incubation with recombinant human uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A7, UGT1A8, and UGT1A10. Additionally, verbascoside showed weak inhibitory potency against CYP1A2 and CYP1B1 with IC50 values of 83 µM and 86 µM, respectively. Potent antioxidant and low cytotoxic potential were observed. Based on these data, verbascoside does not possess any clinically relevant CYP-mediated interaction potential, but it has effective biological activity. Therefore, verbascoside could be considered as a lead compound for further drug development and as an adjuvant during tuberculosis treatment.

New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.

RATIONALE: The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. METHODS: The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer (pH 7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. RESULTS: Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M - H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. CONCLUSIONS: 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.

Kuopio birth cohort - design of a Finnish joint research effort for identification of environmental and lifestyle risk factors for the wellbeing of the mother and the newborn child.

BACKGROUND: A Finnish joint research effort Kuopio Birth Cohort (KuBiCo) seeks to evaluate the effects of genetics, epigenetics and different risk factors (medication, nutrition, lifestyle factors and environmental aspects) during pregnancy on the somatic and psychological health status of the mother and the child. METHODS: KuBiCo will ultimately include information on 10,000 mother-child pairs who have given their informed consent to participate in this cohort. Identification of foetal health risk factors that can potentially later manifest as disease requires a repository of relevant biological samples and a flexible open up-to-date data handling system to register, store and analyse biological, clinical and questionnaire-based data. KuBiCo includes coded questionnaire-based maternal background data gathered before, during and after the pregnancy and bio-banking of maternal and foetal samples that will be stored in deep freezers. Data from the questionnaires and biological samples will be collected into one electronic database. KuBiCo consists of several work packages which are complementary to each other: Maternal, foetal and placental metabolism and omics; Paediatrics; Mental wellbeing; Prenatal period and delivery; Analgesics and anaesthetics during peripartum period; Environmental effects; Nutrition; and Research ethics. DISCUSSION: This report describes the set-up of the KuBiCo and descriptive analysis from 3532 parturients on response frequencies and feedback to KuBiCo questionnaires gathered from June 2012 to April 2016. Additionally, we describe basic demographic data of the participants (n = 1172). Based on the comparison of demographic data between official national statistics and our descriptive analysis, KuBiCo represents a cross-section of Finnish pregnant women.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-ß-hydroxysteroid dehydrogenase 2 - a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

A liquid chromatography-tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine.

Cocktail phenotyping using specific probe drugs for cytochrome P450 (CYP) enzymes provides information on the real-time activity of multiple CYPs. We investigated different sample preparation techniques and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple protein precipitation for the analysis of nine CYP probe drugs and their metabolites in human serum and urine. Specific CYP probe drugs (melatonin, CYP1A2; nicotine, CYP2A6; bupropion, CYP2B6; repaglinide, CYP2C8; losartan, CYP2C9; omeprazole, CYP2C19 and CYP3A4; dextromethorphan, CYP2D6; chlorzoxazone, CYP2E; midazolam, CYP3A4) and their main metabolites, with the exception of 3'-hydroxyrepaglinide, were quantified in human serum and urine using the developed LC-MS/MS method. The analytical method was fully validated showing high selectivity, linearity, acceptable accuracy (85-115 %) and precision (2-19 %) and applied to a pharmacokinetic study in four healthy volunteers after oral administration of drugs given as a cocktail. All probe drugs and their metabolites (totally 19 analytes) were detected and quantified from human serum and urine over the time range of 1 to 6 h after oral administration. Therefore, the proposed method is applicable for drug interaction and CYP phenotyping studies utilizing a cocktail approach. Graphical Abstract Workflow overwiew of cocktail CYP-phenotyping study.

Triethylenetetramine modulates polyamine and energy metabolism and inhibits cancer cell proliferation.

Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.

Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans.

Critical analysis of carcinogenicity study outcomes. Relationship with pharmacological properties.

Predicting the outcome of life-time carcinogenicity studies in rats based on chronic (6-month) toxicity studies in this species is possible in some instances. This should reduce the number of such studies and hence have a significant impact on the total number of animals used in safety assessment of new medicines. From a regulatory perspective, this should be sufficient to grant a waiver for a carcinogenicity study in those cases where there is confidence in the outcome of the prediction. Pharmacological properties are a frequent key factor for the carcinogenic mode of action of some pharmaceuticals, but data-analysis on a large dataset has never been formally conducted. We have conducted an analysis of a dataset based on the perspective of the pharmacology of 255 compounds from industrial and regulatory sources. It is proposed that a pharmacological, class-specific, model may consist of an overall causal relationship between the pharmacological class and the histopathology findings in rats after 6 months treatment, leading to carcinogenicity outcome after 2 years. Knowledge of the intended drug target and pathway pharmacology should enhance the prediction of either positive or negative outcomes of rat carcinogenicity studies. The goal of this analysis is to review the pharmacological properties of compounds together with the histopathology findings from the chronic toxicity study in rodents in order to introduce an integrated approach to estimate the risk of human carcinogenicity of pharmaceuticals. This approach would allow scientists to define conditions under which 2-year rat carcinogenicity studies will or will not add value to such an assessment. We have demonstrated the possibility of a regulatory waiver for a carcinogenicity study in rats, as currently discussed in the International Council for Harmonization (ICH) - formerly known as the International Conference on Harmonization (ICH), by applying the proposed prediction approach in a number of case studies.

Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin via human CYP2A6 and its mouse and pig orthologous enzymes.

1. Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods. 2. In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19. 3. 6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (Km: 0.64-0.91 µM; Vmax: 0.81-0.89 min(-1)) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10 µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species. 4. Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5. 5. These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.

Transcriptomic Analysis of Human Primary Bronchial Epithelial Cells after Chloropicrin Treatment.

Chloropicrin is a vaporizing toxic irritant that poses a risk to human health if inhaled, but the mechanism of its toxicity in the respiratory tract is poorly understood. Here, we exposed human primary bronchial epithelial cells (HBEpC) to two concentrations of chloropicrin (10-50 µM) for 6 or 48 h and used genomic microarray, flow cytometry, and TEM-analysis to monitor cellular responses to the exposures. The overall number of differentially expressed transcripts with a fold-change > ± 2 compared to controls increased with longer exposure times. The initial response was activation of genes with a higher number of up- (512 by 10 µM and 408 by 40 µM chloropicrin) rather than down-regulated transcripts (40 by 10 µM and 215 by 40 µM chloropicrin) at 6 h seen with both exposure concentrations. The number of down-regulated transcripts, however, increased with the exposure time. The differentially regulated transcripts were further examined for enriched Gene Ontology Terms (GO) and KEGG-pathways. According to this analysis, the "ribosome" and "oxidative phosphorylation" were the KEGG-pathways predominantly affected by the exposure. The predominantly affected (GO) biological processes were "protein metabolic process" including "translation," "cellular protein complex assembly," and "response to unfolded protein." Furthermore, the top pathways, "NRF2-activated oxidative stress" and "Ah-receptor signaling," were enriched in our data sets by IPA-analysis. Real time qPCR assay of six selected genes agreed with the microarray analysis. In addition, chloropicrin exposure increased the numbers of late S and/or G2/M-phase cells as analyzed by flow cytometry and induced autophagy as revealed by electron microscopy. The targets identified are critical for vital cellular functions reflecting acute toxic responses and are potential causes for the reduced viability of epithelial cells after chloropicrin exposure.

Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine.

There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1-M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4-M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4-M7) that could make humans more susceptible to lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.

In silico prediction of the site of oxidation by cytochrome P450 3A4 that leads to the formation of the toxic metabolites of pyrrolizidine alkaloids.

In humans, the metabolic bioactivation of pyrrolizidine alkaloids (PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation of their necine bases at C3 or C8 of heliotridine- and retronecine-type PAs or at the N atom of the methyl substituent of otonecine-type PAs. However, no attempts have been made to identify which C atom is the most favorable site for hydroxylation in silico. Here, in order to determine the site of hydroxylation that eventually leads to the formation of the toxic metabolites produced from lasiocarpine, retrorsine, and senkirkin, we utilized the ligand-based electrophilic Fukui function f(-)(r) and hydrogen-bond dissociation energies (BDEs) as well as structure-based molecular docking. The ligand-based computations revealed that the C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of senkirkin were chemically the most susceptible locations for electrophilic oxidizing reactions. Similarly, according to the predicted binding orientation in the active site of the crystal structure of human CYP3A4 (PDB code: 4I4G ), the alkaloids were positioned in such a way that the C3 atom of lasiocarpine and retrorsine and the C26 of senkirkin were closest to the catalytic heme Fe. Thus, it is concluded that the C3 atom of lasiocarpine and retrorsine and C26 of senkirkin are the most favored sites of hydroxylation that lead to the production of their toxic metabolites.

Applying human and pig hepatic in vitro experiments for sulfur mustard study: screening and identification of metabolites by liquid chromatography/tandem mass spectrometry.

RATIONALE: Sulfur mustard is a chemical warfare agent (CWA) with high toxicity and complex metabolism. This study aimed at identification of new metabolic biomarkers for sulfur mustard using in in vitro exposures and various mass spectrometric techniques. METHODS: Human and pig liver subcellular fractions were used as biocatalysts. Metabolites were screened by liquid chromatography and tandem mass spectrometry (LC/MS/MS) using positive electrospray ionization (ESI). For structural identification, product ion scans (MS/MS, MS(3) ) and accurate mass measurements using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) were acquired. RESULTS: Sulfur mustard is metabolized in vitro by S-oxidation and glutathione (GSH) conjugations. One S-oxidized metabolite, bis(2-chloroethyl) sulfoxide (m/z 175), was formed in both species only when liver microsomes were present in incubations, and it was the main metabolite if GSH was not added into the reaction mixture. However, conjugation with GSH was found to be a spontaneous reaction in physiological pH and buffered solution. Three GSH conjugates of sulfur mustard were detected and identified, among which two were novel; 2-((2-(S-glutathionyl)ethyl)thio)ethanol (m/z 412) and 2-((2-(S-glutathionyl)ethyl)thio)ethyl phosphate (m/z 492). CONCLUSIONS: To our knowledge, this was the first time that S-oxidized metabolites and GSH conjugates of sulfur mustard have been detected and identified from human samples in vitro by LC/MS/MS. The usefulness of the GSH conjugates to serve as biomarkers for sulfur mustard exposure in human samples requires further studies.

Analysis of nitromethane from samples exposed in vitro to chloropicrin by stable isotope dilution headspace gas chromatography with mass spectrometry.

Chloropicrin (trichloronitromethane) is a widely used soil fumigant and an old chemical warfare agent. The metabolism of chloropicrin is not well known in mammals but nitromethane has been shown to be one of its main metabolites. Here, a fast and simple headspace gas chromatography with mass spectrometry method was applied for the measurement of nitromethane from aqueous samples. The analytical method was validated using stable isotope labeled internal standard and a small sample volume of 260 µL. No conventional sample preparation steps were needed. The method was accurate (relative standard deviations ≤1.5%) and linear (R(2) = 0.9996) within the concentration range of 0.1-6.0 µg/mL. This method was used to measure nitromethane in in vitro incubations with human and pig liver cell fractions containing enzymes for xenobiotic metabolism, exposed to chloropicrin. The results indicate that the presence of glutathione is necessary for the formation of nitromethane from chloropicrin. Also, nitromethane was formed mostly in liver cytosol fractions, but not in microsomal fractions after the incubation with chloropicrin. Our results suggest that although nitromethane is not the unequivocal biomarker of chloropicrin exposure, this method could be applied for screening the elevated levels in humans after chloropicrin exposure.

Identification of a new reactive metabolite of pyrrolizidine alkaloid retrorsine: (3H-pyrrolizin-7-yl)methanol.

Pyrrolizidine alkaloids (PAs) such as retrorsine are common food contaminants that are known to be bioactivated by cytochrome P450 enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites known as dehydropyrrolizidine alkaloids (DHPs). We compared how both electrochemical (EC) and human liver microsomal (HLM) oxidation of retrorsine could produce short-lived intermediate metabolites; we also characterized a toxicologically important metabolite, (3H-pyrrolizin-7-yl)methanol. The EC cell was coupled online or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas the HLM oxidation was performed in 100 mM potassium phosphate (pH 7.4) in the presence of NADPH at 37 °C. The EC cell oxidation of retrorsine produced 12 metabolites, including dehydroretrorsine (m/z 350, [M + H(+)]), which was degraded to a new reactive metabolite at m/z 136 ([M + H(+)]). The molecular structure of this small metabolite was determined using high-resolution mass spectrometry and NMR spectroscopy followed by chemical synthesis. In addition, we also identified another minor but reactive metabolite at m/z 136, an isomer of (3H-pyrrolizin-7-yl)methanol. Both (3H-pyrrolizin-7-yl)methanol and its minor isomer were also observed after HLM oxidation of retrorsine and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the presence of reduced glutathione (GSH), each isomer formed identical GSH conjugates at m/z 441 and m/z 730 in the negative ESI-MS. Because (3H-pyrrolizine-7-yl)methanol) and its minor isomer subsequently reacted with GSH, it is concluded that (3H-pyrrolizin-7-yl)methanol may be a common toxic metabolite arising from PAs.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.