A remnant planetary core in the hot-Neptune desert.

The interiors of giant planets remain poorly understood. Even for the planets in the Solar System, difficulties in observation lead to large uncertainties in the properties of planetary cores. Exoplanets that have undergone rare evolutionary processes provide a route to understanding planetary interiors. Planets found in and near the typically barren hot-Neptune 'desert'1,2 (a region in mass-radius space that contains few planets) have proved to be particularly valuable in this regard. These planets include HD149026b3, which is thought to have an unusually massive core, and recent discoveries such as LTT9779b4 and NGTS-4b5, on which photoevaporation has removed a substantial part of their outer atmospheres. Here we report observations of the planet TOI-849b, which has a radius smaller than Neptune's but an anomalously large mass of [Formula: see text] Earth masses and a density of [Formula: see text] grams per cubic centimetre, similar to Earth's. Interior-structure models suggest that any gaseous envelope of pure hydrogen and helium consists of no more than [Formula: see text] per cent of the total planetary mass. The planet could have been a gas giant before undergoing extreme mass loss via thermal self-disruption or giant planet collisions, or it could have avoided substantial gas accretion, perhaps through gap opening or late formation6. Although photoevaporation rates cannot account for the mass loss required to reduce a Jupiter-like gas giant, they can remove a small (a few Earth masses) hydrogen and helium envelope on timescales of several billion years, implying that any remaining atmosphere on TOI-849b is likely to be enriched by water or other volatiles from the planetary interior. We conclude that TOI-849b is the remnant core of a giant planet.

Are Environmental Levels of Nonsteroidal Anti-Inflammatory Drugs a Reason for Concern? Chronic Life-Cycle Effects of Naproxen in Zebrafish.

The nonsteroidal anti-inflammatory drug naproxen (NPX) is among the most consumed pharmaceuticals worldwide, being detected in surface waters within the ng to µg/L range. Considering the limited chronic ecotoxicity data available for NPX in aquatic ecosystems, the present study aimed at evaluating its impact in the model organism Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (0.1 to 5.0 µg/L). An integration of apical endpoints, i.e., survival, growth, and reproduction, with gonad histopathology and gene transcription (RNA-seq) was performed to provide additional insights into the mode of action (MoA) of NPX. NPX decreased zebrafish growth and reproduction and led to histopathological alterations in gonads at concentrations as low as 0.1 µg/L. At the molecular level, 0.7 µg/L of NPX led to a disruption in gonads transcription of genes involved in several biological processes associated with reproduction, mainly involving steroid hormone biosynthesis and epigenetic/epitranscriptomic machineries. Collectively, these results show that environmentally realistic concentrations of NPX affect zebrafish reproduction and associated signaling pathways, indicating that current hazard and risk assessment data for NPX underestimate the environmental risk of this pharmaceutical.

Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations.

The antidiabetic drug Metformin (MET), one of the most prevalent pharmaceuticals in the environment, is currently detected in surface waters in the range of ng/L to low µg/L. As current knowledge regarding the long-term effects of environmentally relevant concentrations of MET in nontarget organisms is limited, the present study aimed at investigating the generational effects of MET, in concentrations ranging from 390 to 14 423 ng/L in the model organism Danio rerio (up to 9 mpf), including the effects on its nonexposed offspring (until 60 dpf). We integrate several apical end points, i.e., embryonic development, survival, growth, and reproduction, with qRT-PCR and RNA-seq analyses to provide additional insights into the mode of action of MET. Reproductive-related parameters in the first generation were particularly sensitive to MET. MET parental exposure impacted critical molecular processes involved in the metabolism of zebrafish males, which in turn affected steroid hormone biosynthesis and upregulated male vtg1 expression by 99.78- to 155.47-fold at 390 and 14 432 MET treatment, respectively, pointing to an estrogenic effect. These findings can potentially explain the significant decrease in the fertilization rate and the increase of unactivated eggs. Nonexposed offspring was also affected by parental MET exposure, impacting its survival and growth. Altogether, these results suggest that MET, at environmentally relevant concentrations, severely affects several biological processes in zebrafish, supporting the urgent need to revise the proposed Predicted No-Effect Concentration (PNEC) and the Environmental Quality Standard (EQS) for MET.

Functional, biochemical and molecular impact of sediment plumes from deep-sea mining on Mytilus galloprovincialis under hyperbaric conditions.

The deep-sea is the biggest ecosystem in the world and despite the extreme conditions that characterize it, is highly biodiverse and complex. Deep-sea mining has been foreseen as a potential and concerning new stressor, and among the deep-sea mining associated stressors, sediment plumes, likely to be released into the water column as a side effect of mining, can reach habitats within a radius of more than a hundred kilometers. The present study examined the effects of suspended sediments of different grain sizes (63-125 µm, 125-250 µm and 250-500 µm) in the model species Mytilus galloprovincialis, at 4 bar, as a proxy to address the potential effects of sediment plumes, in the water column, with different grain sizes under high pressure conditions. Functional (filtration rate - FR), biochemical (catalase - CAT, glutathione s-transferase - GST, lipid peroxidation - LPO) and molecular (gene expression of [actin (ACTN), glutathione S-transferase alpha (GSTA), superoxide dismutase 2 (SOD2), catalase (CAT), heat shock protein 60 (HSP60), cytochrome c oxidase (COI) and DNA mismatch repair protein (MSH6)]) endpoints were studied in juvenile organisms. The FR decreased significantly for all tested grain size ranges, with a more severe effect for the particles with a diameter between 63 and 125 µm. In addition to the FR, significant changes were also observed for all tested biomarkers. Gene expression was significantly downregulated for CAT and ACTN. Overall, this study demonstrated that the smaller sized particles are the ones leading to more severe effects. Given their high dispersion potential and longer suspension periods under mining operation scenarios, particular attention should be given to the release of sediment plumes that may affect deep-sea environments and the water column. It is, therefore, vital to create standards and guidelines for sustainable mining practices.

The anti-lipidemic drug simvastatin modifies epigenetic biomarkers in the amphipod Gammarus locusta.

The adverse effects of certain environmental chemicals have been recently associated with the modulation of the epigenome. Although changes in the epigenetic signature have yet to be integrated into hazard and risk assessment, they are interesting candidates to link environmental exposures and altered phenotypes, since these changes may be passed across multiple non-exposed generations. Here, we addressed the effects of simvastatin (SIM), one of the most prescribed pharmaceuticals in the world, on epigenetic regulation using the amphipod Gammarus locusta as a proxy, to support its integration into hazard and environmental risk assessment. SIM is a known modulator of the epigenome in mammalian cell lines and has been reported to impact G. locusta ecological endpoints at environmentally relevant levels. G. locusta juveniles were exposed to three SIM environmentally relevant concentrations (0.32, 1.6 and 8 µg L-1) for 15 days. Gene transcription levels of selected epigenetic regulators, i.e., dnmt1, dmap1, usp7, kat5 and uhrf1 were assessed, along with the quantification of DNA methylation levels and evaluation of key ecological endpoints: survival and growth. Exposure to 0.32 and 8 µg L-1 SIM induced significant downregulation of DNA methyltransferase 1 (dnmt1), concomitant with global DNA hypomethylation and growth impacts. Overall, this work is the first to validate the basal expression of key epigenetic regulators in a keystone marine crustacean, supporting the integration of epigenetic biomarkers into hazard assessment frameworks.

Chronic exposure to environmentally relevant levels of simvastatin disrupts zebrafish brain gene signaling involved in energy metabolism.

Simvastatin (SIM), a hypocholesterolaemic drug belonging to the statins group, is a widely prescribed pharmaceutical for prevention of cardiovascular diseases. Several studies showed that lipophilic statins, as SIM, cross the blood-brain barrier and interfere with the energy metabolism of the central nervous system in humans and mammalian models. In fish and other aquatic organisms, the effects of SIM on the brain energy metabolism are unknown, particularly following exposure to low environmentally relevant concentrations. Therefore, the present study aimed at investigating the influence of SIM on gene signaling pathways involved in brain energy metabolism of adult zebrafish (Danio rerio) following chronic exposure (90 days) to environmentally relevant SIM concentrations ranging from 8 ng/L to 1000 ng/L. Real-time PCR was used to determine the transcript levels of several genes involved in different pathways of the brain energy metabolism (glut1b, gapdh, acadm, accα, fasn, idh3a, cox4i1, and cox5aa). The findings here reported integrated well with ecological and biochemical responses obtained in a parallel study. Data demonstrated that SIM modulates transcription of key genes involved in the mitochondrial electron transport chain, in glucose transport and metabolism, in fatty acid synthesis and ß-oxidation. Further, SIM exposure led to a sex-dependent transcription profile for some of the studied genes. Overall, the present study demonstrated, for the first time, that SIM modulates gene regulation of key pathways involved in the energy metabolism in fish brain at environmentally relevant concentrations.

Chronic effects of triclocarban in the amphipod Gammarus locusta: Behavioural and biochemical impairment.

Triclocarban (TCC), a common antimicrobial agent widely used in many household and personal care products, has been widely detected in aquatic ecosystems worldwide. Due to its high lipophilicity and persistence in the aquatic ecosystems, TCC is of emerging environmental concern. Despite the frequently reported detection of TCC in the environment and significant uncertainties about its long term effects on aquatic ecosystems, few studies have addressed the chronic effects of TCC in aquatic organisms at ecologically relevant concentrations. Therefore, we aimed at testing a broad range of biological responses in the amphipod Gammarus locusta following a chronic (60 days) exposure to environmentally relevant concentrations of TCC (100, 500 and 2500ng/L). This work integrated biochemical markers of oxidative stress (catalase (CAT), glutathione-s-transferase (GST) and lipid peroxidation (LPO)) and neurotransmission (acetylcholinesterase (AChE)) with several key ecological endpoints, i.e. behaviour, survival, individual growth and reproduction. Significant alterations were observed in all biochemical markers. While AChE showed a dose-response curve (with a significant increased activity at a TCC concentration of 2500ng/L), oxidative stress markers did not follow a dose-response curve, with significant increase at 100 and/or 500ng/L and a decreased activity in the highest concentration (2500ng/L). The same effect was observed in the females' behavioural response, whereas males' behaviour was not affected by TCC exposure. The present study represents a first approach to characterize the hazard of TCC to crustaceans.

Worldwide burden of gastric cancer in 2010 attributable to high sodium intake in 1990 and predicted attributable burden for 2030 based on exposures in 2010.

Assessing the impact that patterns of Na intake may have on gastric cancer will provide a more comprehensive estimation of Na reduction as a primary prevention approach. We aimed to estimate the proportion of gastric cancer cases that are attributable to Na intake above the recommendation by the WHO (≤2 g/d) throughout the world in 2010, as well as expected values for 2030. Population attributable fractions (PAF) were computed for 187 countries, using Na intakes in 1990 and 2010 and estimates of the association between Na intake and gastric cancer, assuming a time lag of 20 years. Median PAF ranged from 10·1% in low to 22·5 % in very high Human Development Index (HDI) countries in men (P<0·001) and from 7·2 to 16·6 %, respectively, among women (P<0·001). An increase in median PAF until 2030 is expected in most settings, except for countries classified as low HDI, in both sexes. High Na intakes account for a large proportion of gastric cancer cases, and proportions are expected to increase in almost all of the countries. Intensified efforts to diminish Na intake in virtually all populations are needed to further reduce gastric cancer burden.

Using zebrafish embryo bioassays to identify chemicals modulating the regulation of the epigenome: a case study with simvastatin.

Contaminants of emerging concern have been increasingly associated with the modulation of the epigenome, leading to potentially inherited and persistent impacts on apical endpoints. Here, we address the performance of the OECD Test No. 236 FET (fish embryo acute toxicity) in the identification of chemicals able to modulate the epigenome. Using zebrafish (Danio rerio) embryos, acute and chronic exposures were performed with the pharmaceutical, simvastatin (SIM), a widely prescribed hypocholesterolemic drug reported to induce inter and transgenerational effects. In the present study, the epigenetic effects of environmentally relevant concentrations of SIM (from 8 ng/L to 2000 ng/L) were addressed following (1) an acute embryo assay based on OECD Test No. 236 FET, (2) a chronic partial life-cycle exposure using adult zebrafish (90 days), and (3) F1 embryos obtained from parental exposed animals. Simvastatin induced significant effects in gene expression of key epigenetic biomarkers (DNA methylation and histone acetylation/deacetylation) in the gonads of exposed adult zebrafish and in 80 hpf zebrafish embryos (acute and chronic parental intergenerational exposure), albeit with distinct effect profiles between biological samples. In the chronic exposure, SIM impacted particularly DNA methyltransferase genes in males and female gonads, whereas in F1 embryos SIM affected mostly genes associated with histone acetylation/deacetylation. In the embryo acute direct exposure, SIM modulated the expression of both genes involved in DNA methylation and histone deacetylase. These findings further support the use of epigenetic biomarkers in zebrafish embryos in a high throughput approach to identify and prioritize epigenome-modulating chemicals.

Exploring psychopathy traits on intertemporal decision-making, neurophysiological correlates, and emotions on time estimation in community adults.

There are certain characteristics of psychopathy that may be related to changes in intertemporal choices. Specifically, traits such as impulsivity or lack of inhibitory control may be associated with a more pronounced discounting function in intertemporal choices (IC) and, in turn, this function may be based on changes in the basic mechanisms of time estimation (TE). Therefore, this study aimed to examine potential differences in neurophysiological correlates, specifically through N1, P3, and LPP measurements, which may be related to TE and IC, examining their modulation according to psychopathic traits, different emotional conditions, and different decision-making conditions. This experimental study included 67 adult participants (48 women) from the northern region of Portugal, who performed an intertemporal decision-making task and, of those, 19 participants (16 women), with a mean age of 25 years (SD = 5.41) and a mean of 16 years of schooling (SD = 3.37) performed the time estimation task. The instruments/measures applied were MoCA, used as a neurocognitive screening tool; the Triarchic Psychopathy Measure (TriPM), a self-report instrument with 58 items that map the core features of psychopathy along three facets - boldness, meanness, and disinhibition - and considers them continuously distributed among the general population; intertemporal decision-making and time estimation tasks - for the time estimation task, the stimuli consisted of 45 color images extracted from the Nencki Affective Picture System (NAPS). In the TE task, there was an almost significant effect of disinhibition on the values of θ, with higher values on this variable associated with greater values of θ in the unpleasant emotional condition. In the IC task, there were no significant effects of any psychopathy measure on the values of the gains and losses ratios. In addition, the analysis of the neurophysiological correlates of the IC task did not reveal a main effect of the decision-making condition, nor effects of any psychopathy measure on the N1 and P3 amplitudes. The analysis of the neurophysiological correlates of the TE task revealed that higher meanness values are associated with smaller N1 amplitude in the pleasant emotional condition, whereas higher disinhibition values are associated with greater N1 amplitude in the pleasant emotional condition. Still in this task, higher disinhibition values were associated with a smaller LPP amplitude in the unpleasant emotional condition. The increase in the distribution of attention resources towards time and/or the increase in activation states, including those originated by responses to emotional stimuli, may be the main factor that alters the way impulsive individuals and, presumably, individuals with high psychopathy, consider time when making decisions.

Metformin disrupts Danio rerio metabolism at environmentally relevant concentrations: A full life-cycle study.

Metformin (MET), an anti-diabetic pharmaceutical of large-scale consumption, is increasingly detected in surface waters. However, current knowledge on the long-term effects of MET on non-target organisms is limited. The present study aimed to investigate the effects of MET in the model freshwater teleost Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (390 to 14 423 ng/L). Considering that the mode of action (MoA) of MET on non-target organisms remains underexplored and that MET may act through similar human pathways, i.e., lipid and energy metabolisms, biochemical markers were used to determine cholesterol and triglycerides levels, as well as mitochondrial complex I activity in zebrafish liver. Also, the hepatosomatic index as an indication of metabolic disruption, and the expression levels of genes involved in MET's putative MoA, i.e. acaca, acadm, cox5aa, idh3a, hmgcra, prkaa1, were determined, the last by qRT-PCR. A screening of mRNA transcripts, associated with lipid and energy metabolisms, and other signaling pathways potentially involved in MET-induced toxicity were also assessed using an exploratory RNA-seq analysis. The findings here reported indicate that MET significantly disrupted critical biochemical and molecular processes involved in zebrafish metabolism, such as cholesterol and fatty acid biosynthesis, mitochondrial electron transport chain and tricarboxylic acid cycle, concomitantly to changes on the hepatosomatic index. Likewise, MET impacted other relevant pathways mainly associated with cell cycle, DNA repair and steroid hormone biosynthesis, here reported for the first time in a non-target aquatic organism. Non-monotonic dose response curves were frequently detected in biochemical and qRT-PCR data, with higher effects observed at 390 and 2 929 ng/L MET treatments. Collectively, the results suggest that environmentally relevant concentrations of MET severely disrupt D. rerio metabolism and other important biological processes, supporting the need to revise the proposed environmental quality standard (EQS) and predicted no-effect concentration (PNEC) for MET.

A compositional link between rocky exoplanets and their host stars.

Stars and planets both form by accreting material from a surrounding disk. Because they grow from the same material, theory predicts that there should be a relationship between their compositions. In this study, we search for a compositional link between rocky exoplanets and their host stars. We estimate the iron-mass fraction of rocky exoplanets from their masses and radii and compare it with the compositions of their host stars, which we assume reflect the compositions of the protoplanetary disks. We find a correlation (but not a 1:1 relationship) between these two quantities, with a slope of >4, which we interpret as being attributable to planet formation processes. Super-Earths and super-Mercuries appear to be distinct populations with differing compositions, implying differences in their formation processes.

Transcriptomic data on the transgenerational exposure of the keystone amphipod Gammarus locusta to simvastatin.

The use of transcriptomics data brings new insights and works as a powerful tool to explore the molecular mode of action (MoA) of transgenerational inheritance effects of contaminants of emerging concern. Therefore, in this dataset, we present the transcriptomic data of the transgenerational effects of environmentally relevant simvastatin levels, one of the most prescribed human pharmaceuticals, in the keystone amphipod species Gammarus locusta. In summary, G. locusta juveniles were maintained under simvastatin exposure up to adulthood (exposed group - F0E) and the offspring of F0E were transferred to control water for the three subsequent generations (transgenerational group - F1T, F2T and F3T). To gain insights into the biological functions and canonical pathways transgenerationally disrupted by simvastatin, a G. locusta de novo transcriptome assembly was produced and the transcriptomic profiles of three individual G. locusta females, per group, over the four generations (F0 to F3) - solvent control groups (F0.C, F1.C, F2.C and F3.C), F0 320 ng/L simvastatin exposed group (F0.320E) and F1 to F3 320 transgenerational group (F1.320T; F2.320T and F3.320T) - were analyzed. Briefly, Illumina HiSeq™ 2500 platform was used to perform RNA sequencing, and due to the unavailability of G. locusta genome, the RNA-seq datasets were assembled de novo using Trinity and annotated with Trinotate software. After assembly and post-processing steps, 106093 transcripts with N50 of 2371 bp and mean sequence length of 1343.98 bp was produced. BUSCO analyses showed a transcriptome with gene completeness of 97.5 % Arthropoda library profile. The Bowtie2, RSEM and edgeR tools were used for the differential gene expression (DEGs) analyses that allowed the identification of a high quantity of genes differentially expressed in all generations. Finally, to identify the main metabolic pathways affected by the transgenerational effects of SIM across all generations, the DGEs genes were blasted onto KEGG pathways database using the KAAS webserver. The data furnished in this article allows a better molecular understanding of the transgenerational effects produced by simvastatin in the keystone amphipod G. locusta and has major implications for hazard and risk assessment of pharmaceuticals and other emerging contaminants. This article is related to the research article entitled "Transgenerational inheritance of chemical-induced signature: a case study with simvastatin [1].

Ecotoxicology of deep-sea environments: Functional and biochemical effects of suspended sediments in the model species Mytilus galloprovincialis under hyperbaric conditions.

The deep-sea is the biggest ecosystem in the world and is characterized by extreme conditions such as high pressure, low temperatures and absence or limited light. Despite the scarce studies due to inaccessibility, these ecosystems are considered highly biodiverse. The deep-sea is subjected to anthropogenic stressors with deep-sea mining being a likely new form of disruption. Understanding how it affects the surrounding environments is paramount to develop guidelines to protect sensitive habitats and allow for responsible exploitation of resources. One of the potential stressors associated with deep-sea mining are the sediment laden plumes that can be generated during the mining process. The present study examined, for the first time, the effects of suspended sediments (0, 1, 2 and 4 g/L) in the model mussel species, Mytilus galloprovincialis, under hyperbaric conditions (1, 4 and 50 Bar). Functional endpoints, i.e. feeding assays, together with biochemical biomarkers of oxidative stress [catalase (CAT), lipid peroxidation (LPO), glutathione-s-transferase (GST) and superoxide dismutase (SOD)] were studied in juvenile mussels. The filtration rate (FR) of M. galloprovincialis decreased with the increment in the sediment concentrations, for all tested pressure conditions (1, 4 and 50 Bar). Significant alterations were also observed for all tested biomarkers, being sediment and pressure-dependent. Interestingly, pressure had an effect in GST activity, that increased in the 4 and 50 Bar experiments in comparison with the results at 1 Bar. Remarkably, filtration rates were significantly affected by pressure. These findings will support the filling of the knowledge gaps related with the hazard assessment of deep-sea mining associated stressors.

Chronic environmentally relevant levels of simvastatin disrupt embryonic development, biochemical and molecular responses in zebrafish (Danio rerio).

Simvastatin (SIM), a hypocholesterolaemic compound, is among the most prescribed pharmaceuticals for cardiovascular disease prevention worldwide. Several studies have shown that acute exposure to SIM causes multiple adverse effects in aquatic organisms. However, uncertainties still remain regarding the chronic effects of SIM in aquatic ecosystems. Therefore, the present study aimed to investigate the effects of SIM in the model freshwater teleost zebrafish (Danio rerio) following a chronic exposure (90 days) to environmentally relevant concentrations ranging from 8 ng/L to 1000 ng/L. This study used a multi-parameter approach integrating distinct ecologically-relevant endpoints, i.e. survival, growth, reproduction and embryonic development, with biochemical markers (cholesterol and triglycerides). Real Time PCR was used to analyse the transcription levels of key genes involved in the mevalonate pathway (hmgcra, cyp51, and dhcr7). Globally, SIM induced several effects that did not follow a dose-response relationship; embryonic development, biochemical and molecular markers, were significantly impacted in the lower concentrations, 8 ng/L, 40 ng/L and/or 200 ng/L, whereas no effects were recorded for the highest tested SIM levels (1000 ng/L). Taken together, these findings expand our understanding of statin effects in teleosts, demonstrating significant impacts at environmentally relevant concentrations and highlight the importance of addressing the effects of chemicals under chronic low-level concentrations.

Accretion-induced variability links young stellar objects, white dwarfs, and black holes.

The central engines of disc-accreting stellar-mass black holes appear to be scaled down versions of the supermassive black holes that power active galactic nuclei. However, if the physics of accretion is universal, it should also be possible to extend this scaling to other types of accreting systems, irrespective of accretor mass, size, or type. We examine new observations, obtained with Kepler/K2 and ULTRACAM, regarding accreting white dwarfs and young stellar objects. Every object in the sample displays the same linear correlation between the brightness of the source and its amplitude of variability (rms-flux relation) and obeys the same quantitative scaling relation as stellar-mass black holes and active galactic nuclei. We also show that the most important parameter in this scaling relation is the physical size of the accreting object. This establishes the universality of accretion physics from proto-stars still in the star-forming process to the supermassive black holes at the centers of galaxies.

The redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cells.

BACKGROUND: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. RESULTS: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. CONCLUSIONS: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.