I have been course leader for Introduction to environmental chemistry (KZ4007), and have lectures in Introduction to environmental chemistry (KZ4007), Toxicology for environmental scientists (MI7015) and Contaminant analysis (MI7019).

My research interests concern the development of target and non-target analysis focusing on high resolution mass spectrometry based adductomics, and its applicability in addressing important challenges in human and environmental health. I have published my work in reputed journals such as Environmental Science & Technology, Chemical Research in Toxicology and Scientific Reports and presented at numerous national and international conferences. My publications list is accessible via my Google Scholar profile.

A selection from Stockholm University publication database

• DNA Adductomics for the Biological Effect Assessment of Contaminant Exposure in Marine Sediments

  2023. Giulia Martella (et al.). Environmental Science and Technology 57 (29), 10591-10603

  Article

  Exposure to chemical pollution can induce genetic andepigeneticalterations, developmental changes, and reproductive disorders, leadingto population declines in polluted environments. These effects aretriggered by chemical modifications of DNA nucleobases (DNA adducts)and epigenetic dysregulation. However, linking DNA adducts to thepollution load in situ remains challenging, and thelack of evidence-based DNA adductome response to pollution hampersthe development and application of DNA adducts as biomarkers for environmentalhealth assessment. Here, we provide the first evidence for pollutioneffects on the DNA modifications in wild populations of Baltic sentinelspecies, the amphipod Monoporeia affinis. A workflow based on high-resolution mass spectrometry to screenand characterize genomic DNA modifications was developed, and itsapplicability was demonstrated by profiling DNA modifications in theamphipods collected in areas with varying pollution loads. Then, thecorrelations between adducts and the contaminants level (polycyclicaromatic hydrocarbons (PAHs), trace metals, and pollution indices)in the sediments at the collection sites were evaluated. A total of119 putative adducts were detected, and some (5-me-dC, N-6-me-dA, 8-oxo-dG, and dI) were structurally characterized. The DNAadductome profiles, including epigenetic modifications, differed betweenthe animals collected in areas with high and low contaminant levels.Furthermore, the correlations between the adducts and PAHs were similaracross the congeners, indicating possible additive effects. Also,high-mass adducts had significantly more positive correlations withPAHs than low-mass adducts. By contrast, correlations between theDNA adducts and trace metals were stronger and more variable thanfor PAHs, indicating metal-specific effects. These associations betweenDNA adducts and environmental contaminants provide a new venue forcharacterizing genome-wide exposure effects in wild populations andapply DNA modifications in the effect-based assessment of chemicalpollution. DNA adductome analysis identifiesexposure to environmentalcontaminants in a sentinel species in the Baltic Sea.

  Read more about DNA Adductomics for the Biological Effect Assessment of Contaminant Exposure in Marine Sediments

• Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis

  2023. Giulia Martella (et al.). Chemical Research in Toxicology 36 (9), 1471-1482

  Article

  Adductomics studies are used for the detection and characterizationof various chemical modifications (adducts) of nucleic acids and proteins.The advancements in liquid chromatography coupled with high-resolutiontandem mass spectrometry (HRMS/MS) have resulted in efficient methodsfor qualitative and quantitative adductomics. We developed an HRMS-basedmethod for the simultaneous analysis of RNA and DNA adducts in a singlerun and demonstrated its application using Baltic amphipods, usefulsentinels of environmental disturbances, as test organisms. The noveltyof this method is screening for RNA and DNA adducts by a single injectionon an Orbitrap HRMS instrument using full scan and data-independentacquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adductsbased on the characteristic neutral loss of ribonucleosides and 2 & PRIME;-deoxyribonucleosides,respectively. In the amphipods, in addition to the nearly 150 putativeDNA adducts characterized earlier, we detected 60 putative RNA adducts.For the structural identification of the detected RNA adducts, theMODOMICS database was used. The identified RNA adducts included simplemono- and dimethylation and other larger functional groups on differentribonucleosides and deaminated product inosine. However, 54 of theseRNA adducts are not yet structurally identified, and further workon their characterization may uncover new layers of information relatedto the transcriptome and help understand their biological significance.Considering the susceptibility of nucleic acids to environmental factors,including pollutants, the developed multi-adductomics methodologywith further advancement has the potential to provide biomarkers fordiagnostics of pollution effects in biota.

  Read more about Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis

• Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry

  2022. Javier Zurita (et al.). Toxics 10 (1)

  Article

  Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

  Read more about Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry

• Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts: Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors

  2022. Efstathios Vryonidis (et al.). Chemical Research in Toxicology 35 (12), 2227-2240

  Article

  Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level. 

  Read more about Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts

• nLossFinder—A Graphical User Interface Program for the Nontargeted Detection of DNA Adducts

  2021. Pedro F.M. Sousa (et al.). Toxics 9 (4)

  Article

  DNA adductomics is a relatively new omics approach aiming to measure known and unknown DNA modifications, called DNA adducts. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has become the most common method for analyzing DNA adducts. Recent advances in the field of mass spectrometry have allowed the possibility to perform a comprehensive analysis of adducts, for instance, by using a nontargeted data-independent acquisition method, with multiple precursor m/z windows as an inclusion list. However, the generated data are large and complex, and there is a need to develop algorithms to simplify and automate the time-consuming manual analysis that has hitherto been used. Here, a graphical user interface (GUI) program was developed, with the purpose of tracking a characteristic neutral loss reaction from tandem mass spectrometry of the nucleoside adducts. This program, called nLossFinder, was developed in the MATLAB platform, available as open-source code. Calf thymus DNA was used as a model for method optimization, and the overall adductomics approach was applied to DNA from amphipods (Monoporeia affinis) collected within the Swedish National Marine Monitoring Program. In the amphipod DNA, over 150 putative adducts were found in comparison to 18 using a manual approach in a previous study. The developed program can improve the processing time for large MS data, as it processes each sample in a few seconds, and hence can be applicable for high-throughput screening of adducts.

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• DNA as an in vitro trapping agent for detection of bulky genotoxic metabolites

  2020. Hitesh V. Motwani. Journal of chromatography. B 1152

  Article

  The instability of electrophilic reactive metabolites in in vitro metabolism studies makes their accurate analysis challenging. To stabilise the reactive compounds prior to their analysis, different trapping agents, such as thiols, amines and cob(I)alamin, have earlier been tested depending on the metabolites to be analysed and the type of study. In the present work, DNA is introduced as a trapping agent for measuring the formation of bulky electrophilic metabolites. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), was used as a model compound in a rat liver S9 metabolic system. Under physiological incubation conditions, B[a]P metabolises to diol epoxide (BPDE) metabolites which were trapped by DNA resulting in the formation of covalently bound DNA adducts. The methodology for analysis of these adducts included extraction of the DNA from the metabolic system, digestion of the DNA to yield nucleosides and analysis of the BPDE-adduct to deoxyguanosine (BPDE-dG) by liquid chromatography coupled to high resolution mass spectrometry (HRMS). The chromatographic conditions in combination with the high mass accuracy data (±3 ppm) was useful in resolving BPDE-dG in its protonated form from the complex set of ions present in the metabolic matrix. The method was validated in terms of sensitivity, specificity, accuracy, precision and recovery, and applied to provide a preliminary estimate of BPDE-dG levels from the metabolism of B[a]P in rat S9. The use of DNA as a trapping agent for in vitro metabolites has a potential to aid in cancer risk assessment procedure of PAHs, for instance, in inter-species comparison of metabolism to reactive metabolites and can be adapted for screening of genotoxic metabolites, e.g., from emerging environmental contaminants.

  Read more about DNA as an in vitro trapping agent for detection of bulky genotoxic metabolites

• DNA epigenetic marks are linked to embryo aberrations in amphipods

  2020. Elena Gorokhova (et al.). Scientific Reports 10

  Article

  Linking exposure to environmental stress factors with diseases is crucial for proposing preventive and regulatory actions. Upon exposure to anthropogenic chemicals, covalent modifications on the genome can drive developmental and reproductive disorders in wild populations, with subsequent effects on the population persistence. Hence, screening of chemical modifications on DNA can be used to provide information on the probability of such disorders in populations of concern. Using a high-resolution mass spectrometry methodology, we identified DNA nucleoside adducts in gravid females of the Baltic amphipods Monoporeia affinis, and linked the adduct profiles to the frequency of embryo malformations in the broods. Twenty-three putative nucleoside adducts were detected in the females and their embryos, and eight modifications were structurally identified using high-resolution accurate mass data. To identify which adducts were significantly associated with embryo malformations, partial least squares regression (PLSR) modelling was applied. The PLSR model yielded three adducts as the key predictors: methylation at two different positions of the DNA (5-methyl-2′-deoxycytidine and N⁶-methyl-2′-deoxyadenosine) representing epigenetic marks, and a structurally unidentified nucleoside adduct. These adducts predicted the elevated frequency of the malformations with a high classification accuracy (84%). To the best of our knowledge, this is the first application of DNA adductomics for identification of contaminant-induced malformations in field-collected animals. The method can be adapted for a broad range of species and evolve as a new omics tool in environmental health assessment.

  Read more about DNA epigenetic marks are linked to embryo aberrations in amphipods

• Serum albumin adducts, DNA adducts and micronuclei frequency measured in benzo[a]pyrene-exposed mice for estimation of genotoxic potency

  2020. Hitesh Motwani (et al.). Mutation research. Genetic toxicology and environmental mutagenesis 849

  Article

  The environmental and food contaminant, benzo[a]pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)}, is classified as a human carcinogen by the International Agency for Research on Cancer. The carcinogenicity of B[a]P is linked to the formation of electrophilic metabolites, namely B[a]P-diol epoxides (BPDEs) occurring as stereoisomers. In this work, we quantified the metabolic formation of BPDE isomers and the genotoxic effect in B[a]P-exposed mice, with an aim to estimate the genotoxic potency of B[a]P per in vivo dose of its most potent metabolite [i.e. ( + )-anti-BPDE]. The increase in frequency of micronuclei ((MN) in erythrocytes was measured as a biomarker for genotoxic effect. Covalent adducts to serum albumin (SA) and those to DNA from the BPDEs were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS), as adducts to histidine (BPDE-His-Pro) and deoxyguanosine (BPDE-dG), respectively. For the first time in animal experiments it was possible to resolve adducts to SA from ( + )-anti-, (-)-anti- and ( +/- )-syn-BPDE isomers by LC-MS/MS. The adduct levels in the protein were about 16 fmol/mg SA, which was orders of magnitude lower than that in the nucleic acid, 28 pmol/mg DNA, in mice exposed to 100 mg B[a]P per kg body weight (bw). Using SA adduct levels, the in vivo dose of ( + )-anti-BPDE was calculated to be approximately 50 nM.h per mg B[a]P per kg bw. This allowed to make a preliminary estimate of the genotoxic potency as 2 parts per thousand fMN per mu M.h of ( + )-anti-BPDE. This estimate was compared to that from another food toxicant, glycidol, studied with similar methods, which indicated that the BPDE has several orders of magnitude higher genotoxic potency. The demonstrated approach on integrating biomarkers of internal dose of a causative agent and that of genotoxic effect for assessing genotoxic potency, using B[a]P as a model, has a potential for improving cancer risk assessment procedures for PAHs.

  Read more about Serum albumin adducts, DNA adducts and micronuclei frequency measured in benzo[a]pyrene-exposed mice for estimation of genotoxic potency

• Solvent-Assisted Paper Spray Ionization Mass Spectrometry (SAPSI-MS) for the Analysis of Biomolecules and Biofluids

  2019. Nicoló Riboni (et al.). Scientific Reports 9

  Article

  Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

  Read more about Solvent-Assisted Paper Spray Ionization Mass Spectrometry (SAPSI-MS) for the Analysis of Biomolecules and Biofluids

• Reaction kinetic studies for comparison of mutagenic potency between butadiene monoxide and glycidamide

  2018. Hitesh Motwani (et al.). Chemico-Biological Interactions 288, 57-64

  Article

  DNA adducts can be formed from covalent binding of electrophilic reactive compounds to the nucleophilic Nand O-atoms of the biomolecule. The O-sites on DNA, with nucleophilic strength (n) of ca. 2, is recognized as a critical site for mutagenicity. Characterization of the reactivity of electrophilic compounds at the O-sites can be used to predict their mutagenic potency in relative terms. In the present study, reaction kinetic experiments were performed for butadiene monoxide (BM) in accordance with the Swain-Scott relation using model nucleophiles representing N- and O-sites on DNA, and earlier for glycidamide (GA) using a similar approach. The epoxide from the kinetic experiments was trapped by cob(I)alamin, resulting in formation of an alkylcobalamin which was analyzed by liquid chromatography tandem mass spectrometry. The Swain-Scott relationship was used to determine selectivity constant (s) of BM and GA as 0.86 and 1.0, respectively. The rate constant for the reaction at n of 2 was extrapolated to 0.023 and 0.038M(-1) h(-1) for BM and GA, respectively, implying a higher mutagenic potency per dose unit of GA compared to BM. The reaction kinetic parameters associated with mutagenic potency were also estimated by a density functional theory approach, which were in accordance to the experimental determined values. These types of reaction kinetic measures could be useful in development of a chemical reactivity based prediction tool that could aid in reduction of animal experiments in cancer risk assessment procedures for relative mutagenicity.

  Read more about Reaction kinetic studies for comparison of mutagenic potency between butadiene monoxide and glycidamide

• Parallelogram based approach for in vivo dose estimation of genotoxic metabolites in humans with relevance to reduction of animal experiments

  2017. Hitesh V. Motwani, Cecilia Frostne, Margareta Törnqvist. Scientific Reports 7

  Article

  When employing metabolism studies of genotoxic compounds/metabolites and cancer tests for risk estimation, low exposure doses in humans are roughly extrapolated from high exposure doses in animals. An improvement is to measure the in vivo dose, i.e. area under concentration-time curve (AUC), of the causative genotoxic agent. In the present work, we propose and evaluate a parallelogram based approach for estimation of the AUC of genotoxic metabolites that incorporates in vitro metabolic data and existing knowledge from published in vivo data on hemoglobin (Hb) adduct levels, using glycidamide (GA) as a case study compound that is the genotoxic metabolite of acrylamide (AA). The estimated value of AUC of GA per AUC of AA from the parallelogram approach vs. that from Hb adduct levels measured in vivo were in good agreement; 0.087 vs. 0.23 in human and 1.4 vs. 0.53 in rat, respectively. The described parallelogram approach is simple, and can be useful to provide an approximate estimation of the AUC of metabolites in humans at low exposure levels for which sensitive methods for analyzing the metabolites are not available, as well as aid in reduction of animal experiments for metabolism studies that are to be used for cancer risk assessment.

  Read more about Parallelogram based approach for in vivo dose estimation of genotoxic metabolites in humans with relevance to reduction of animal experiments

• Interaction of benzo[a] pyrene diol epoxide isomers with human serum albumin: Site specific characterisation of adducts and associated kinetics

  2016. Hitesh V. Motwani, Emelie Westberg, Margareta Törnqvist. Scientific Reports 6

  Article

  Carcinogenicity of benzo[a] pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)} involves DNA-modification by B[a] P diol epoxide (BPDE) metabolites. Adducts to serum albumin (SA) are not repaired, unlike DNA adducts, and therefore considered advantageous in assessment of in vivo dose of BPDEs. In the present work, kinetic experiments were performed in relation to the dose (i.e. concentration over time) of different BPDE isomers, where human SA (hSA) was incubated with respective BPDEs under physiological conditions. A liquid chromatography (LC) tandem mass spectrometry methodology was employed for characterising respective BPDE-adducts at histidine and lysine. This strategy allowed to structurally distinguish between the adducts from racemic anti-and syn-BPDE and between (+)- and (-)-anti-BPDE, which has not been attained earlier. The adduct levels quantified by LC-UV and the estimated rate of disappearance of BPDEs in presence of hSA gave an insight into the reactivity of the diol epoxides towards the N-sites on SA. The structure specific method and dosimetry described in this work could be used for accurate estimation of in vivo dose of the BPDEs following exposure to B[a] P, primarily in dose response studies of genotoxicity, e.g. in mice, to aid in quantitative risk assessment of PAHs.

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• Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions

  2016. Jenny Aasa (et al.). Mutation research. Genetic toxicology and environmental mutagenesis 805, 38-45

  Article

  Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration x cell treatment time. Gly induced mutations in the hprt-gene at ante of 0.08 +/- 0:01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5 rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

  Read more about Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions

• Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples

  2015. Henrik Carlsson (et al.). Chemical Research in Toxicology 28 (11), 2120-2129

  Article

  Electrophiles have the ability to form adducts to nudeophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 +/- 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using L-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 degrees C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life <1 min. Parallel experiments with AA and MVK showed that the two vinyl ketones react approximately 2 x 10(3) times faster than AA. The EVK-Hb adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening.

  Read more about Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples

• Alkylcobyrinate from sucralose and mechanistic aspects of its Co-C bond cleavage

  2014. Hitesh V. Motwani (et al.). Tetrahedron Letters 55 (16), 2667-2670

  Article

  Reaction of heptamethyl cob(I)yrinate with 1',6'-dichloro-1',6'-dideoxy-beta-D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside (sucralose) gave an alkylcobyrinate, heptamethyl-6'-[alpha-D-galactopyranoside- 1'-chloro-1',6'-dideoxy-beta-D-fructofuranosyl-4-chloro-4-deoxy]cobyrinate perchlorate, [(Suc)Cob(III) 7C(1)ester]ClO4. Cleavage behavior of Co-C bond of the alkylcobyrinate was investigated under various conditions (hv, H+ and reduction) using UV-vis spectroscopy combined with the EPR spin-trapping technique, and by cyclic voltammetry.

  Read more about Alkylcobyrinate from sucralose and mechanistic aspects of its Co-C bond cleavage

• In vivo doses of butadiene epoxides as estimated from in vitro enzyme kinetics by using cob(I)alamin and measured hemoglobin adducts: An inter-species extrapolation approach

  2014. Hitesh Motwani, Margareta Törnqvist. Toxicology and Applied Pharmacology 281 (3), 276-284

  Article

  1,3-Butadiene (BD) is a rodent and human carcinogen. In the cancer tests, mice have been much more susceptible than rats with regard to BD-induced carcinogenicity. The species-differences are dependent on metabolic formation/disappearance of the genotoxic BD epoxy-metabolites that lead to variations in the respective in vivo doses, i.e. "area under the concentration-time curve" (AUC). Differences in AUC of the most gentoxic BD epoxy-metabolite, diepoxybutane (DEB), are considered important with regard to cancer susceptibility. The present work describes: the application of cob(I)alamin for accurate measurements of in vitro enzyme kinetic parameters associated with BD epoxy-metabolites in human, mouse and rat; the use of published data on hemoglobin (Hb) adduct levels of BD epoxides from BD exposure studies on the three species to calculate the corresponding AUCs in blood; and a parallelogram approach for extrapolation of AUC of DEB based on the in vitro metabolism studies and adduct data from in vivo measurements. The predicted value of AUC of DEB for humans from the parallelogram approach was 0.078 nM . h for 1 ppm . h of BD exposure compared to 0.023 nM . h/ppm . h as calculated from Hb adduct levels observed in occupational exposure. The corresponding values in nM . h/ppm . h were for mice 41 vs. 38 and for rats 126 vs. 137 from the parallelogram approach vs. experimental exposures, respectively, showing a good agreement This quantitative inter-species extrapolation approach will be further explored for the clarification of metabolic rates/pharmacokinetics and the AUC of other genotoxic electrophilic compounds/metabolites, and has a potential to reduce and refine animal experiments.

  Read more about In vivo doses of butadiene epoxides as estimated from in vitro enzyme kinetics by using cob(I)alamin and measured hemoglobin adducts

• Cob(I)alamin as a Quantitative Tool for Analysis, Metabolism and Toxicological Studies of Electrophilic Compounds: Butadiene Epoxides, Glycidamide and Sucralose

  2011. Hitesh Vijay Motwani.

  Thesis (Doc)

  Vitamin B₁₂ can be reduced to cob(I)alamin [Cbl(I)], which is one of the most powerful nucleophiles known and referred to as a “supernucleophile”. Cbl(I) was applied as a tool in toxicological studies of the air pollutant 1,3-butadiene (BD), the toxicant acrylamide (AA) present in many foods, and the artificial sweetener sucralose.

  BD, a human carcinogen, is metabolised to genotoxic epoxides, two monoepoxides and the most potent diepoxybutane (DEB). AA, classified as a probable human carcinogen, is metabolised to the genotoxic epoxide glycidamide (GA). Due to their reactivity, quantitative analysis of the epoxides presents an analytical challenge. By using Cbl(I) for trapping, a sensitive and accurate method to quantify the epoxides as alkylcobalamins by LC-MS/MS in metabolism studies was developed and validated.

  Using the Cbl(I) method, enzyme kinetic parameters, V_max and K_m, were determined for the metabolic steps associated with the BD epoxides and with the formation of GA from AA, in liver S9 fractions of human, mouse and rat.

  An approach to estimate dose in vivo (i.e. area under concentration time curve, AUC) of BD epoxides by scaling the enzyme kinetic data was designed. The AUCs obtained from in vitro were evaluated by comparing with AUCs in vivo that were calculated from published haemoglobin adduct data. The AUCs from in vitro and in vivo showed to be in agreement with each other for mouse and rat, and this evaluation allowed prediction of the unknown AUC of DEB in human from BD exposure. This approach has a potential to reduce animal experiments in the future.

  Sucralose is of concern due to its chlorinated structure and persistence in the aquatic environment. It was demonstrated that Cbl(I) reacts with sucralose, also under in vitro physiological conditions, which might have toxicological significance. The demonstrated reaction also suggested a potential role for Cbl(I) in dehalogenation/degradation of sucralose. This was evaluated and shown possible using heptamethyl cobyrinate, a model compound for cobalamin.

  Read more about Cob(I)alamin as a Quantitative Tool for Analysis, Metabolism and Toxicological Studies of Electrophilic Compounds

• Cob(I)alamin reacts with sucralose to afford an alkylcobalamin: Relevance to in vivo cobalamin and sucralose interaction

  2011. Hitesh V. Motwani (et al.). Food and Chemical Toxicology 49 (4), 750-757

  Article

  Vitamin B(12), viz., cyano- or hydroxo-cobalamin, can be chemically or enzymatically converted into the derivatives methyl- and adenosyl-cobalamin, which are complex organometallic cofactors associated with several cobalamin-dependent enzymes. The reduced form of vitamin B(12), cob(I)alamin {Cbl(I)}, obtained by reduction of hydroxocobalamin (OH-Cbl) with e.g. sodium borohydride, is one of the most powerful nucleophiles known. Cbl(I) was shown to react readily with the synthetic sweetener sucralose (1,6-dichloro-1,6-dideoxy-β-d-fructofuranosyl-4-chloro-4-deoxy-α-d-galactopyranoside) in an aqueous system to form an alkylcobalamin (Suc-Cbl). This occurred by replacement of one of the three chlorine atoms of sucralose with a cobalamin moiety. The efficiency of trapping sucralose in presence of excess Cbl(I) was estimated to be >90%. Furthermore, in an in vitro study using human liver S9 with NADPH regeneration, in presence of OH-Cbl and sucralose, Suc-Cbl was shown to be formed. The Suc-Cbl was characterized primarily by LC-ESI(+)-MS/MS. Given the human consumption of sucralose from food and beverages, such a reaction between the sweetener and reduced vitamin B(12) could occur in vivo.

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• A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts

  2010. Ronnie Davies (et al.). Chemical Research in Toxicology 23 (3), 540-546

  Article

  Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)-carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization tinder the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

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• Cob(I)alamin for trapping butadiene epoxides in metabolism with rat S9 and for determining associated kinetic parameters

  2009. Hitesh V Motwani (et al.). Chemical Research in Toxicology 22 (9), 1509-1516

  Article

  The reduced state of vitamin B(12), cob(I)alamin, acts as a supernucleophile that reacts ca. 10(5) times faster than standard nucleophiles, for example, thiols. Methods have been developed for trapping electrophilically reactive compounds by exploiting this property of cob(I)alamin. 1,3-Butadiene (BD) has recently been classified as a group 1 human carcinogen by the International Agency for Research on Cancer (IARC). The carcinogenicity of BD is considered to be dependent on the activation or deactivation of the reactive metabolites of BD, that is, the epoxides (oxiranes) 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol). Cytochrome P450 (P450) isozymes are involved in oxidation of BD to EB and further activation to DEB. EB and DEB are hydrolyzed by epoxide hydrolases (EH) to 3,4-dihydroxy-1-butene (BDdiol) and EBdiol, respectively. EBdiol can also be formed by oxidation of BDdiol. In the present study, cob(I)alamin was used for instant trapping of the BD epoxide metabolites generated in in vitro metabolism to study enzyme kinetics. The substrates EB, DEB, and BDdiol were incubated with rat S9 liver fraction, and apparent K(m) and apparent V(max), were determined. The ratio of conversion of EB to DEB (by P450) to the rate of deactivation of DEB by EH was 1.09. Formation of EBdiol from hydrolysis of DEB was ca. 10 times faster than that from oxidation of BDdiol. It was also found that the oxidation of EB to DEB was much faster than that of BDdiol to EBdiol. The study offers comparative enzyme kinetic data of different BD metabolic steps, which is useful for quantitative interspecies comparison. Furthermore, a new application of cob(I)alamin was demonstrated for the measurement of enzyme kinetics of compounds that form electophilically reactive metabolites.

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• Reductive dechlorination of sucralose mediated by vitamin B12 derivatives

  2008. Hitesh Motwani (et al.).

  Conference

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• Cob(I)alamin as an analytical tool to study 1,3-butadiene metabolism in human, mouse and rat S9 liver fractions

  2007. Hitesh Motwani (et al.). Gordon Research Conference on Vitamin B12 and Corphins

  Conference

  Read more about Cob(I)alamin as an analytical tool to study 1,3-butadiene metabolism in human, mouse and rat S9 liver fractions

Show all publications by Hitesh Vijay Motwani at Stockholm University