T2-FLAIR Mismatch: An Imaging Biomarker for Children's MYB/MYBL1-Altered Diffuse Astrocytoma or Angiocentric Glioma.

BACKGROUND AND PURPOSE: T2-FLAIR mismatch is a highly specific imaging biomarker of IDH-mutant diffuse astrocytoma in adults. It has however also been described in MYB/MYBL1-altered low grade tumors. Our aim was to assess the diagnostic power of the T2-FLAIR mismatch in IDH-mutant astrocytoma and MYB/MYBL1-altered low-grade tumors in children and correlate this mismatch with histology. MATERIALS AND METHODS: We evaluated MR imaging examinations of all pediatric patients, performed at the Princess Máxima Center for Pediatric Oncology and the University Medical Center Utrecht between January 2012 and January 2023, with the histomolecular diagnosis of IDH-mutant astrocytoma, diffuse astrocytoma MYB/MYBL1-altered, or angiocentric glioma, and the presence of T2-FLAIR mismatch was assessed. Histologically, the presence of microcysts in the tumor (a phenomenon suggested to be correlated with T2-FLAIR mismatch in IDH-mutant astrocytomas in adults) was evaluated. RESULTS: Nineteen pediatric patients were diagnosed with either IDH-mutant astrocytoma (n = 8) or MYB/MYBL1-altered tumor (n = 11: diffuse astrocytoma, MYB- or MYBL1-altered n = 8; or angiocentric glioma n = 3). T2-FLAIR mismatch was present in 11 patients, 3 (38%) in the IDH-mutant group and 8 (73%) in the MYB/MYBL1 group. No correlation was found between T2-FLAIR mismatch and the presence of microcysts or an enlarged intercellular space in either IDH-mutant astrocytoma (P = .38 and P = .56, respectively) or MYB/MYBL1-altered tumors (P = .36 and P = .90, respectively). CONCLUSIONS: In our pediatric population, T2-FLAIR mismatch was more often found in MYB/MYBL1-altered tumors than in IDH-mutant astrocytomas. In contrast to what has been reported for IDH-mutant astrocytomas in adults, no correlation was found with microcystic changes in the tumor tissue. This finding challenges the hypothesis that such microcystic changes and/or enlarged intercellular spaces in the tissue of these tumors are an important part of explaining the occurrence of the T2-FLAIR mismatch.

Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Ultra-fast deep-learned CNS tumour classification during surgery.

Central nervous system tumours represent one of the most lethal cancer types, particularly among children1. Primary treatment includes neurosurgical resection of the tumour, in which a delicate balance must be struck between maximizing the extent of resection and minimizing risk of neurological damage and comorbidity2,3. However, surgeons have limited knowledge of the precise tumour type prior to surgery. Current standard practice relies on preoperative imaging and intraoperative histological analysis, but these are not always conclusive and occasionally wrong. Using rapid nanopore sequencing, a sparse methylation profile can be obtained during surgery4. Here we developed Sturgeon, a patient-agnostic transfer-learned neural network, to enable molecular subclassification of central nervous system tumours based on such sparse profiles. Sturgeon delivered an accurate diagnosis within 40 minutes after starting sequencing in 45 out of 50 retrospectively sequenced samples (abstaining from diagnosis of the other 5 samples). Furthermore, we demonstrated its applicability in real time during 25 surgeries, achieving a diagnostic turnaround time of less than 90 min. Of these, 18 (72%) diagnoses were correct and 7 did not reach the required confidence threshold. We conclude that machine-learned diagnosis based on low-cost intraoperative sequencing can assist neurosurgical decision-making, potentially preventing neurological comorbidity and avoiding additional surgeries.

A case report of a blueberry muffin baby caused by congenital self-healing indeterminate cell histiocytosis.

BACKGROUND: Blueberry muffin is a descriptive term for a neonate with multiple purpuric skin lesions. Many causes are known, amongst them life-threatening diseases like congenital infections or leukemia. Indeterminate cell histiocytosis (ICH) is an exceptionally rare cause of blueberry muffin rash. ICH is a histiocytic disorder which can be limited to the skin or can present with systemic involvement. A mutation that has been described in histiocytic disorders is a MAP2K1 mutation. In ICH, this mutation has previously been described in merely one case. CASE PRESENTATION: A term male neonate was admitted to the neonatology ward directly after birth because of a blueberry muffin rash. ICH was diagnosed on skin biopsy. The lesions resolved spontaneously. The patient is currently 3 years old and has had no cutaneous lesions or systemic involvement so far. This disease course is similar to that of the Hashimoto-Pritzker variant of LCH. CONCLUSIONS: ICH can manifest in neonates as resolving skin lesions. It is limited to the skin in most cases, but systemic development is possible. Therefore, it is essential to confirm the diagnosis with a biopsy before the lesions resolve and to monitor these patients closely with routine follow-up.

A personally guided tour on some of our data with the Ames assay-A tribute to Professor Bruce Ames.

In contributing to this Special Issue of Mutation Research dedicated to Professor Bruce N. Ames in recognition of his 90th birthday in December 2018, we intend to portray the importance not only of the Ames Salmonella/mammalian-microsome mutagenicity assay in some of our studies over the years, but also the importance of the insight that Bruce Ames brought to the field of genetic toxicology.

A crystallographic study of crystalline casts and pseudomorphs from the 3.5†Ga Dresser Formation, Pilbara Craton (Australia).

Crystallography has a long history of providing knowledge and methods for applications in other disciplines. The identification of minerals using X-ray diffraction is one of the most important contributions of crystallography to earth sciences. However, when the crystal itself has been dissolved, replaced or deeply modified during the geological history of the rocks, diffraction information is not available. Instead, the morphology of the crystal cast provides the only crystallographic information on the original mineral phase and the environment of crystal growth. This article reports an investigation of crystal pseudomorphs and crystal casts found in a carbonate-chert facies from the 3.48 Ga-old Dresser Formation (Pilbara Craton, Australia), considered to host some of the oldest remnants of life. A combination of X-ray microtomography, energy-dispersive X-ray spectroscopy and crystallographic methods has been used to reveal the original phases of these Archean pseudomorphs. It is found with a high degree of confidence that the original crystals forming in Archean times were hollow aragonite, the high-temperature polymorphs of calcium carbonate, rather than other possible alternatives such as gypsum (CaSO4·2H20) and nahcolite (NaHCO3). The methodology used is described in detail.

Complex patterns in fossilized stromatolites revealed by hyperspectral imaging (400-2496 nm).

Hyperspectral imaging (400-2496 nm) was used to quantitatively map surface textures and compositional variations in stromatolites to determine whether complexity of textures could be used as evidence to support biogenicity in the absence of preserved biomarkers. Four samples of 2.72-2.4 Ga stromatolites from a variety of settings, encompassing marine and lacustrine environments, were selected for hyperspectral imaging. Images of the sawn surfaces of samples were processed to identify reflectance and mineral absorption features and quantify their intensity (as an index of mineral abundance) using automated feature extraction. Amounts of ferrous iron were quantified using a ratio of reflectance at 1650 and 1299 nm. Visible near infrared imagery (400-970 nm) did not reveal additional textural patterns to those obtained from visual inspection. Shortwave infrared imagery (1000-2496 nm), however, revealed complex laminar and convoluted patterns, including a distinctive texture of sharp peaks and broad, low troughs in one sample, similar to living tufted microbial mats. Spectral analysis revealed another sample to be composed of dolomite. Two other samples were dominated by calcite or chlorite ± illite. Large variations in amounts of ferrous iron were found, but ferric iron was exclusively located in the oxidation crust. Hyperspectral imaging revealed large differences between parts of a sample of biogenic and non-biogenic origin. The former was characterized by calcite with varying amounts of ferrous iron, distributed in lenticular, convoluted patterns; the latter by Mg-Fe chlorite with large amounts of aluminium silicate, distributed as fine laminar layers. All minerals identified by hyperspectral imaging were confirmed by thin section petrography and XRD analyses. Spatial statistics generated from quantitative minerals maps showed different patterns between these different parts of the sample. Thus, hyperspectral imaging was shown to be a powerful tool for detecting structures in stromatolites that could be used, together with other lines of evidence, to support biogenicity.

Lithostratigraphic analysis of a new stromatolite-thrombolite reef from across the rise of atmospheric oxygen in the Paleoproterozoic Turee Creek Group, Western Australia.

This study describes a previously undocumented dolomitic stromatolite-thrombolite reef complex deposited within the upper part (Kazput Formation) of the c. 2.4-2.3 Ga Turee Creek Group, Western Australia, across the rise of atmospheric oxygen. Confused by some as representing a faulted slice of the younger c. 1.8 Ga Duck Creek Dolomite, this study describes the setting and lithostratigraphy of the 350-m-thick complex and shows how it differs from its near neighbour. The Kazput reef complex is preserved along 15 km of continuous exposure on the east limb of a faulted, north-west-plunging syncline and consists of 5 recognisable facies associations (A-E), which form two part regressions and one transgression. The oldest facies association (A) is characterised by thinly bedded dololutite-dolarenite, with local domical stromatolites. Association B consists of interbedded columnar and stratiform stromatolites deposited under relatively shallow-water conditions. Association C comprises tightly packed columnar and club-shaped stromatolites deposited under continuously deepening conditions. Clotted (thrombolite-like) microbialite, in units up to 40 m thick, dominates Association D, whereas Association E contains bedded dololutite and dolarenite, and some thinly bedded ironstone, shale and black chert units. Carbon and oxygen isotope stratigraphy reveals a narrow range in both δ(13) Ccarb values, from -0.22 to 0.97‰ (VPDB: average = 0.68‰), and δ(18) O values, from -14.8 to -10.3‰ (VPDB), within the range of elevated fluid temperatures, likely reflecting some isotopic exchange. The Kazput Formation stromatolite-thrombolite reef complex contains features of younger Paleoproterozoic carbonate reefs, yet is 300-500 Ma older than previously described Proterozoic examples worldwide. Significantly, the microbial fabrics are clearly distinct from Archean stromatolitic marine carbonate reefs by way of containing the first appearance of clotted microbialite and large columnar stromatolites with complex branching arrangements. Such structures denote a more complex morphological expression of growth than previously recorded in the geological record and may link to the rise of atmospheric oxygen.

A Paleoarchean coastal hydrothermal field inhabited by diverse microbial communities: the Strelley Pool Formation, Pilbara Craton, Western Australia.

The 3.4-Ga Strelley Pool Formation (SPF) at the informally named 'Waterfall Locality' in the Goldsworthy greenstone belt of the Pilbara Craton, Western Australia, provides deeper insights into ancient, shallow subaqueous to possibly subaerial ecosystems. Outcrops at this locality contain a thin (<3 m) unit of carbonaceous and non-carbonaceous cherts and silicified sandstones that were deposited in a shallow-water coastal environment, with hydrothermal activities, consistent with the previous studies. Carbonaceous, sulfide-rich massive black cherts with coniform structures up to 3 cm high are characterized by diverse rare earth elements (REE) signatures including enrichment of light [light rare earth elements (LREE)] or middle rare earth elements and by enrichment of heavy metals represented by Zn. The massive black cherts were likely deposited by mixing of hydrothermal and non-hydrothermal fluids. Coniform structures in the cherts are characterized by diffuse laminae composed of sulfide particles, suggesting that unlike stromatolites, they were formed dominantly through physico-chemical processes related to hydrothermal activity. The cherts yield microfossils identical to previously described carbonaceous films, small and large spheres, and lenticular microfossils. In addition, new morphological types such as clusters composed of large carbonaceous spheroids (20-40 µm across each) with fluffy or foam-like envelope are identified. Finely laminated carbonaceous cherts are devoid of heavy metals and characterized by the enrichment of LREE. This chert locally contains conical to domal structures characterized by truncation of laminae and trapping of detrital grains and is interpreted as siliceous stromatolite formed by very early or contemporaneous silicification of biomats with the contribution of silica-rich hydrothermal fluids. Biological affinities of described microfossils and microbes constructing siliceous stromatolites are under investigation. However, this study emphasizes how diverse the microbial community in Paleoarchean coastal hydrothermal environment was. We propose the diversity is at least partially due to the availability of various energy sources in this depositional environment including reducing chemicals and sunlight.

Adiponectin and incident coronary heart disease and stroke. A systematic review and meta-analysis of prospective studies.

The plasma concentration of adiponectin, an adipokine that has anti-inflammatory, anti-atherogenic and insulin sensitizing properties, is lower in obese subjects and could therefore be a target for therapy. In order to review and meta-analyse prospective cohort studies investigating adiponectin concentration and the risk for incident coronary heart disease (CHD) or stroke, a systematic search of MEDLINE, EMBASE and Cochrane databases was performed. Two independent reviewers selected prospective cohort studies investigating the relationship between adiponectin level and incident CHD or stroke using 'adiponectin' and 'cardiovascular disease' or 'stroke' and their synonyms, excluding patients with clinically manifest vascular disease. Random-effects models were used to calculate pooled relative risks (RRs) and 95% confidence intervals (95% CI). Generalized least squares regression was used to assess dose-response relationships for adiponectin concentrations from studies that provided RRs solely based upon categorical data regression. In total, 16 prospective cohort studies, comprising 23,919 patients and 6,870 CHD or stroke outcome events, were included in the meta-analyses. An increase of 1 standard deviation in log-transformed adiponectin did not lower the risk for CHD (RR 0.97; 95% CI 0.86-1.09). A 10 µg mL(-1) increase in adiponectin conferred a RR of 0.91 (95% CI 0.80-1.03) for CHD and a RR 1.01 (95% CI 0.97-1.06) for stroke. In conclusion, plasma adiponectin is not related to the risk for incident CHD or stroke.

Characterisation of non-obese diabetic patients with marked insulin resistance identifies a novel familial partial lipodystrophy-associated PPARγ mutation (Y151C).

AIMS/HYPOTHESIS: Familial partial lipodystrophy (FPLD) is a rare metabolic disorder with clinical features that may not be readily recognised. As FPLD patients require a specific therapeutic approach, early identification is warranted. In the present study we aimed to identify cases of FPLD among non-obese patients with type 2 diabetes mellitus and marked insulin resistance. METHODS: We searched the databases of three diabetic outpatient clinics for patients with marked insulin resistance, arbitrarily defined as the use of ≥100 U insulin/day, and BMI ≤ 27 kg/m(2). In all patients, metabolic variables and anthropomorphic measurements were evaluated and DNA was sequenced for mutations in the genes encoding lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC). RESULTS: Out of 5,221 diabetic individuals, 24 patients fulfilled all criteria. Twelve patients were willing to participate, of whom five showed clinical features of lipodystrophy. In three of these patients the clinical diagnosis of FPLD was confirmed by the presence of mutations in LMNA or PPARG; one patient harboured a novel heterozygous mutation (Y151C) in PPARG. The Y151C mutant displayed impaired DNA-binding capacity and hence reduced transcriptional activity compared with wild-type PPARγ. Dominant-negative activity was absent. CONCLUSION/INTERPRETATION: The combination of BMI ≤ 27 kg/m(2) and the use of >100 U insulin/day increases the chance of identifying lipodystrophy. Thus careful assessment of clinical features of FPLD should be considered in these patients, allowing earlier therapeutic interventions.

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression.

Inter-individual variability in cytochrome P450 (CYP)-mediated xenobiotic metabolism is extensive. CYP1A2 is involved in the metabolism of drugs and in the bioactivation of carcinogens. The objective of this study was to functionally characterize eight polymorphic forms of human CYP1A2, namely T83M, S212C, S298R, G299S, I314V, I386F, C406Y and R456H. cDNAs of these variants were constructed and coexpressed in Escherichia coli with human NADPH cytochrome P450 oxidoreductase (CYPOR). All variants showed similar levels of apoprotein and holoprotein expression, except for I386F and R456H, which showed only apoprotein, and both were functionally inactive. The activity of CYP1A2 variants was investigated using 8 substrates, measuring 16 different activity parameters. The resulting heterogeneous activity data set was analyzed together with CYP1A2 wild-type (WT) form, applying multivariate analysis. This analysis indicated that variant G299S is substantially altered in catalytic properties in comparison with WT, whereas variant T83M is slightly but significantly different from the WT. Among CYP1A2 variants, out of the heterogeneous set of eight substrates, carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was the most discriminative compound. In addition, R456 could be identified as an important residue for proper heme binding and stabilization.

Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites.

We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodiumberghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1alphaa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1alphaa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.

Self-assembled silica-carbonate structures and detection of ancient microfossils.

We have synthesized inorganic micron-sized filaments, whose microstucture consists of silica-coated nanometer-sized carbonate crystals, arranged with strong orientational order. They exhibit noncrystallographic, curved, helical morphologies, reminiscent of biological forms. The filaments are similar to supposed cyanobacterial microfossils from the Precambrian Warrawoona chert formation in Western Australia, reputed to be the oldest terrestrial microfossils. Simple organic hydrocarbons, whose sources may also be abiotic and indeed inorganic, readily condense onto these filaments and subsequently polymerize under gentle heating to yield kerogenous products. Our results demonstrate that abiotic and morphologically complex microstructures that are identical to currently accepted biogenic materials can be synthesized inorganically.

Heterologous expression of xenobiotic mammalian-metabolizing enzymes in mutagenicity tester bacteria: an update and practical considerations.

There is an increasing need for metabolic competent cell systems for the mechanistic studies of biotransformation of xenobiotics in toxicology in general and in genotoxicology in particular. These cell systems combine the heterologous expression of a particular mammalian biotransformation enzyme with a specific target/ end point by which a functional analysis of the expressed gene product in the (geno)toxicity of chemicals can be performed. cDNAs of an increasing number of mammalian biotransformation enzymes is being cloned. The construction of specific expression vectors permits their heterologous expression in laboratory bacteria, such as Escherichia coli strains. This development does not only allow biochemical and enzymatic studies of (pure) enzyme preparations but also facilitates the engineering of metabolically competent mutagenicity tester bacteria, thereby providing new tools for genotoxicity testing and for studying of the roles of biotransformation in chemical carcinogenesis. In this review, we describe an update as well as an evaluation of enzymes expressed in mutagenicity tester bacteria. Four types of biotransformation enzymes are now expressed in these bacteria, namely, GSTs, CYPs, NATs, and STs. The expression of these enzymes in the tester bacteria and their subsequent application in mutagenicity assays demonstrates that heterologous expression in this type of bacteria has a number implications for the functionality of the biotransformation enzymes as well as for the functioning of the tester bacteria in mutagenicity detection. We also describe here a number of practical considerations in this regard.

Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies.

We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP-enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems.

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.

Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens.

In this study we describe the development of strain BMX100, a new Escherichia coli K12 tester strain, derived from MX100, a strain which was constructed for detection of mutagens and for mechanistic studies of chemical carcinogens. We demonstrate here that strain BMX100 can be used for stable expression of human CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochrome P450 reductase. Mutagenicity of precarcinogens known to be bioactivated by CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), could be detected. The mutagenic activity of 2-AA using BMX100 expressing CYP1A2 alone and in combination with rat CYP reductase was respectively 10 and 20 times higher than in BMX100 with the standard metabolic activation system, rat liver S9 fraction. Furthermore, the mutagenicity of 2-AA could be nullified by alpha-naphthoflavone, a known inhibitor of CYP1A2. IQ responded equally in BMX100 expressing the CYP1A2-reductase fusion protein as compared with usage of rat liver S9 fraction. Rat liver S9 fraction was much more potent in generating a mutagenic response to AFB1 in BMX100 than in the strain expressing human CYP1A2 alone or CYP1A2 fused to rat reductase. The results described in this study demonstrate that this new E.coli strain can function as a human CYP1A2-competent prokaryotic mutagenicity test system and they seem to characterize BMX100 as a strain of interest for studies to identify individual human CYPs involved in bioactivation and bioinactivation reactions of putative genotoxins.

Characterization of enzyme activities and cofactors involved in bioactivation and bioinactivation of chemical carcinogens in the tester strains Escherichia coli K12 MX100 and Salmonella typhimurium LT2 TA100.

MX100 is an Escherichia coli K12 genotoxicity tester strain, especially developed for mechanistic studies of chemical mutagens and carcinogens. For the study of the role of specific enzymes in the bioactivation and bioinactivation of carcinogens, it is necessary to characterize MX100 as far as its metabolic bio(in)activation capacities are concerned. In this study such a characterization is performed in two types of cell-free lysates, one derived from stationary phase cells, grown in rich medium (SR-lysates) and one from exponentially growing cells (log phase), cultured in minimal medium (LM-lysates). Six Phase I enzyme activities of aromatic NADPH hydroxylase, NADH hydroxylase, flavin-containing monooxygenase (FMO), nitroreductase, DT-diaphorase and NADPH ferredoxin:oxidoreductase were determined. Activities of six Phase II enzymes glutathione S-transferases (GSTs), N-aryl acetyltransferase (NAT), arylamine sulphotransferase, UDP-glucuronyltransferase and epoxide hydratase and of the Phase III enzyme cysteine conjugate beta-lyase were subsequently assessed. In addition, five antioxidant enzymes: superoxide dismutase (SOD), catalase, glutathione (GSH)-reductase, GSH-peroxidase and alkyl hydroperoxide reductase; as well as concentrations of glutathione (GSH) and its disulphide (GSSG), were measured. The activity parameters of all enzymes were compared with those obtained in similar lysates of the Salmonella strain TA100 and in rat liver preparations. The results indicate that MX100 as well as TA100 contain relatively low oxidative but high reductase Phase I activities. Both strains demonstrated low activities for the Phase II conjugation enzymes except for GSTs. In MX100, relatively high activities were detected for all antioxidative enzymes, activities which were lower in TA100. Significant differences in activities were observed between the SR-lysates derived from stationary phase/rich medium and LM-lysates from log phase/minimal medium cells for nitroreductase, GST, SOD, catalase, NADPH ferredoxin:oxidoreductase as well as in GSH content. In general, we described for the first time a metabolic characterization of the E.coli tester strain MX100 and the Salmonella typhimurium strain TA100 and discussed the results in terms of its significance for carcinogen bioactivation and bioinactivation capacities.

MX100, a new Escherichia coli tester strain for use in genotoxicity studies.

The development of a new Escherichia coli tester strain for use in metabolic and mechanistic studies of genotoxins, strain MR2101/pKR11, has recently been reported. This strain, a derivative of the E. coli K12 laboratory strain AB1157, has sensitivity towards the detection of base-substitution mutagenesis, monitored by the reversion of arginine auxotrophy [argE3, (ochre)]. Besides arginine, MK2101/pKR11 is auxotrophic for histidine (hisG4), leucine (leuB6), proline (DeltaproA) and threonine (thr-1). MX100 was developed to overcome the auxotrophy for four amino acids of MR2101/pKR11 which are non-essential for the mutagenic responsiveness of the strain. We restored the biosynthesis for these four amino acids in MR2101/pKR11, resulting in strain MX100. This strain showed an almost 2-fold increase in mutagenic activity relative to MR2101/pKR11 with a set of diagnostic mutagens (aflatoxin B1, benzo[a]pyrene, 4-nitroquinoline-1-oxide, 2,7-dimethyl-benz[a]anthracene and others) and was further characterized with other types of mutagens in which it showed sensitivity towards the detection of oxidative (H2O2t-butyl-hydroperoxide, cumene-hydroperoxide, KO2) and carbonyl mutagens (methylglyoxal, malondialdehyde). As MX100 seems to have the right characteristics of a versatile genotoxicity tester strain and due to the extensive genetic and physiological knowledge of E. coli K12 in general and AB1157 in particular, we propose that MX100 could serve as mother strain for the development of specialized tester strains, of interest in studies of metabolism and/or mechanism of action of genotoxic carcinogens.