Inorganic Mercury Compounds Research Articles

Comparison between GC–MS and GC–ICPMS using isotope dilution for the simultaneous monitoring of inorganic and methyl mercury, butyl and phenyl tin compounds in biological tissues

The aim of this work is to compare simultaneous isotope dilution analysis of organotin and organomercury compounds by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP/MS) on certified bivalve samples. These samples were extracted by microwave with tetramethylammonium hydroxide (TMAH). Derivatization with both NaBEt4 and NaBPr4 was evaluated, and analytical performances were compared. Two CRM materials, BCR-710 and CRM-477, were analyzed by both techniques to verify accuracy. A mixed spike containing (201)Hg-enriched methylmercury (MeHg), (199)Hg-enriched inorganic mercury (iHg), (119)Sn-enriched monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) as well as homemade (116)Sn-enriched monophenyltin (MPT), diphenyltin (DPT), and triphenyltin (TPT) was used for the isotope dilution analysis of samples. The two techniques studied were compared in terms of classic analytical parameters: linearity, precision or repeatability (i.e., percent relative standard deviation, RSD%), limit of detection (LOD), and limit of quantification (LOQ), showing excellent linearity, precision below 12% for all analytes, and LOQs of 0.06-1.45 pg for GC-MS and 0.02-0.27 pg for GC-ICP/MS.

Mercury detoxification using genetic engineered Nicotiana tabacum

Phytoremediation is a low cost alternative solution to soil contamination compared with traditional removal and/or disposal techniques. One of the phytoremediation technologies is the phytovolatilization, whereby the contaminant is not primarily accumulated in above-ground tissues, but is instead transformed by the plant into the atmosphere. The detoxification of highly toxic organmercurial compounds and subsequent volatilization of elemental mercury is a unique example for the successful phytoremediation based on genetic engineering approach. For mercury removal techniques we constructed a dicistronic construct containing the bacterial mercury detoxification genes merA and merB under the control of the Arabidopsis Actin2 promoter and terminator. The resulted construct was introduced to Agrobacterium competent cells using heat shock transformation method. In the mean time, we germinated tobacco (Nicotiana tabacum var. Gold leaf) seeds on MS media and infected leaf discs of 6-8 weeks tobacco seedlings with Agrobacterium containing Ti plasmid harboring merA/merB dicistronic construct. The results showed that about 90% of tobacco seedlings were carrying the mer genes. Tobacco seeds were collected from wild type and transgenic lines and tested for mercury resistance. The results showed that transgenic plants are resistant to both Phenyl Mercuric Acetate (PMA) and HgCl2. The root length and dry weight of wild and transgenic seedlings growing on both media amended with mercury compounds and media without mercury (control) were scored. The results showed that the root lengths and dry weight of the transgenic lines are significantly higher by 60 and 17-folds, respectively, compared to wild type. The results showed clear evidence that the transgenic plants are resistant to both organic and inorganic mercury compounds and can be used to clean up mercury contaminated sites.

Human Exposure and Health Effects of Inorganic and Elemental Mercury

Mercury is a toxic and non-essential metal in the human body. Mercury is ubiquitously distributed in the environment, present in natural products, and exists extensively in items encountered in daily life. There are three forms of mercury, i.e., elemental (or metallic) mercury, inorganic mercury compounds, and organic mercury compounds. This review examines the toxicity of elemental mercury and inorganic mercury compounds. Inorganic mercury compounds are water soluble with a bioavailability of 7% to 15% after ingestion; they are also irritants and cause gastrointestinal symptoms. Upon entering the body, inorganic mercury compounds are accumulated mainly in the kidneys and produce kidney damage. In contrast, human exposure to elemental mercury is mainly by inhalation, followed by rapid absorption and distribution in all major organs. Elemental mercury from ingestion is poorly absorbed with a bioavailability of less than 0.01%. The primary target organs of elemental mercury are the brain and kidney. Elemental mercury is lipid soluble and can cross the blood-brain barrier, while inorganic mercury compounds are not lipid soluble, rendering them unable to cross the blood-brain barrier. Elemental mercury may also enter the brain from the nasal cavity through the olfactory pathway. The blood mercury is a useful biomarker after short-term and high-level exposure, whereas the urine mercury is the ideal biomarker for long-term exposure to both elemental and inorganic mercury, and also as a good indicator of body burden. This review discusses the common sources of mercury exposure, skin lightening products containing mercury and mercury release from dental amalgam filling, two issues that happen in daily life, bear significant public health importance, and yet undergo extensive debate on their safety.

Evaluation of Developmental Toxicants and Signaling Pathways in a Functional Test Based on the Migration of Human Neural Crest Cells

Background: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the U.S. Environmental Protection Agency and others. Because impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC cells is desirable for a DT test battery.Objective: We developed a robust and widely applicable human-relevant NC function assay that would allow for sensitive screening of environmental toxicants and defining toxicity pathways.Methods: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC cells (MINC assay), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways.Results: Methylmercury (50 nM), valproic acid (> 10 µM), and lead-acetate [Pb(CH3CO2)4] (1 µM) affected the migration of NC cells more potently than migration of other cell types. The MINC assay correctly identified the NC toxicants triadimefon and triadimenol. Additionally, it showed different sensitivities to various organic and inorganic mercury compounds. Using the MINC assay and applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC cells.Conclusions: The MINC assay faithfully models human NC cell migration, and it reveals impairment of this function by developmental toxicants with good sensitivity and specificity.

Quercetin protects human-derived liver cells against mercury-induced DNA-damage and alterations of the redox status

Aim of this study was to investigate the cytotoxic and genotoxic properties of inorganic and organic mercury compounds, i.e., HgCl2 and methylmercury (MeHg). In addition, the DNA-protective and antioxidant effects of the flavonoid quercetin (QC) were studied. All experiments were conducted with human-derived liver cells (HepG2), which possess antioxidant and drug-metabolizing enzymes in an inducible form. 8-Hydroxydeoxyguanosine (8-OHdG) and comet formation were monitored as endpoints of DNA damage. The impact of the metal compounds on the redox status was also investigated, since it is assumed that their toxic effects are due to oxidative damage. A number of biochemical parameters related to oxidative stress, namely glutathione, malondialdehyde, protein carbonyl and formation of reactive oxygen species (ROS) were measured after treatment of the cells with the mercury compounds in the presence and absence of quercetin. To elucidate the mechanisms that underlie the effects of QC, three protocols (pre-, simultaneous and post-treatment) were used. Both mercury compounds (range 0.1–5.0μM) caused induction of DNA migration and formation of 8-OHdG. In combination with the flavonoid (range 0.1–5.0μM), DNA-protective effects of QC were observed after pre- and simultaneous treatment but not when the flavonoid was added after treatment with the metal compounds. Exposure to the metal compounds led also to substantial changes of all parameters of the redox status and co-treatment experiments with QC showed that these alterations are reversed by the flavonoid. Taken together, the results of our experiments indicate that these two mercury compounds cause DNA damage and oxidative stress in human-derived liver cells and that the flavonoid reduces these effects. Since the concentrations of the metals and of the flavonoids used in the present work reflect human exposure, our findings can be taken as an indication that QC may protect humans against the adverse effects caused by the metal.

Thermodynamic Study of the Extraction of Inorganic Mercury Compounds in Fish Species of the International Anzali Wetland, Iran

AbstractInorganic mercury concentration was measured in the muscle tissues of four types of fish in the aquatic international Anzali Wetland ecosystem in Guilan. In the same ecosystem, the northern pike is able to concentrate inorganic mercury in its body more than other fish. In each species, a fish with the highest amount of inorganic mercury was selected for determination of thermodynamic parameters of inorganic mercury extraction using calculation of the equilibrium constant and fitting of ln Kc versus inverse temperature at the atmospheric pressure. The origins of thermodynamic parameters were discussed. The extraction processes were done in the range of temperatures from 331.15 to 365.15 K and at atmospheric pressure. Results show that the extraction of mercury compounds from SH groups of sulfhydryl proteins in fish muscle tissue is an endothermic process with a positive value for entropy and Gibbs free energy changes at room temperature.

Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: implications for treatment of mercury poisoning

Mercury toxicity is a highly interesting topic in biomedicine due to the severe endpoints and treatment limitations. Selenite serves as an antagonist of mercury toxicity, but the molecular mechanism of detoxification is not clear. Inhibition of the selenoenzyme thioredoxin reductase (TrxR) is a suggested mechanism of toxicity. Here, we demonstrated enhanced inhibition of activity by inorganic and organic mercury compounds in NADPH-reduced TrxR, consistent with binding of mercury also to the active site selenolthiol. On treatment with 5 μM selenite and NADPH, TrxR inactivated by HgCl(2) displayed almost full recovery of activity. Structural analysis indicated that mercury was complexed with TrxR, but enzyme-generated selenide removed mercury as mercury selenide, regenerating the active site selenocysteine and cysteine residues required for activity. The antagonistic effects on TrxR inhibition were extended to endogenous antioxidants, such as GSH, and clinically used exogenous chelating agents BAL, DMPS, DMSA, and α-lipoic acid. Consistent with the in vitro results, recovery of TrxR activity and cell viability by selenite was observed in HgCl(2)-treated HEK 293 cells. These results stress the role of TrxR as a target of mercurials and provide the mechanism of selenite as a detoxification agent for mercury poisoning.

ChemInform Abstract: Electrode Reactions and Electroanalysis of Organomercury Compounds

AbstractReview: 396 refs.

Cytogenotoxicity in uroepithelial cells of women exposed to mercury in a mining area

ObjectiveTo investigate biomarkers of cytogenotoxicity in women exposed to mercury in a mining area. Mercury has been associated with cytogenotoxicity in human and animal models but has not been considered...

Intoxication par le mercure

Mercury is a widespread heavy metal with potential severe impacts on human health. Exposure conditions to mercury and profile of toxicity among humans depend on the chemical forms of the mercury: elemental or metallic mercury, inorganic or organic mercury compounds. This article aims to reviewing and synthesizing the main knowledge of the mercury toxicity and its organic compounds that clinicians should know. Acute inhalation of metallic or inorganic mercury vapours mainly induces pulmonary diseases, whereas chronic inhalation rather induces neurological or renal disorders (encephalopathy and interstitial or glomerular nephritis) . Methylmercury poisonings from intoxicated food occurred among some populations resulting in neurological disorders and developmental troubles for children exposed in utero. Treatment using chelating agents is recommended in case of symptomatic acute mercury intoxication; sometimes it improves the clinical effects of chronic mercury poisoning. Although it is currently rare to encounter situations of severe intoxication, efforts remain necessary to decrease the mercury concentration in the environment and to reduce risk on human health due to low level exposure (dental amalgam, fish contamination by organic mercury compounds…). In case of occupational exposure to mercury and its compounds, some disorders could be compensated in France. Clinicians should work with toxicologists for the diagnosis and treatment of mercury intoxication.

Roles Played by MerE and MerT in the Transport of Inorganic and Organic Mercury Compounds in Gram-negative Bacteria

In order to clarify the physiological roles played by MerP, MerT and MerE in Gram-negative bacteria, we constructed the plasmids pTP4 and pTPE21, which contained the genes merR, merT and merP, from the Pseudomonas K-62 plasmid pMR26, or the same genes with the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100), respectively. Cells containing pTP4 showed increased hypersensitivity to Hg(II), but maintained a normal sensitivity to CH3Hg(I). However, cells with pTPE21 exhibited increased hypersensitivity to Hg(II) and CH3Hg(I). Cells with pTP4 accumulated appreciably more Hg(II) than control cells, but no significant difference was observed in their uptake of 14CH3Hg(I). In contrast, the cells containing pTPE21 accumulated significantly larger amounts of Hg(II) and 14CH3Hg(I) than either control cells or cells with pTP4. These results suggest that the mer operons have evolved to redirect uptake of mercurials into dedicated, specific and relatively high-affinity transport systems comprising the small periplasmic protein MerP and two inner membrane proteins, MerT and MerE.

Health implications of mercury exposure in children

Mercury is a ubiquitous environmental toxin that can produce a wide range of health effects in humans. It is found in three chemical forms: organic, inorganic and elemental (mercury). The sources of exposure are also markedly different for the different forms of mercury. Diet, especially fish and other seafood, is the main source of exposure of the general public to organic mercury. Dental amalgam is the most important source for elemental mercury vapour in the general population. Inorganic mercury compounds are known as 'mercuric salts', which are sometimes used in skin-lightening creams and as antiseptic creams and ointments. Though its use has been banned in some countries, these products are still available on the world market. Each mercury form possesses a different risk to human health. Mercury can be transferred prenatally to the developing foetus via the placenta or postnatally from breast milk to the nursing infant. Children are potentially more susceptible to mercury than adults due to differences in the stages of brain development and organ growth that occur during the foetal, infant and childhood developmental periods. The objective of this paper is to review the relevant literature concerning transplacental and lactational exposure to mercury, taking into account mercury speciation, in order to have a critical assessment of its adverse health effects. Attention is also given to available studies in Saudi Arabia where skin-lightening creams, dental amalgam and thimerosal are still in use.

Targeted Therapies in Solid Tumours: Pinpointing the Tumours Achilles Heel

It is now exactly 100 years ago (1908) that Paul Ehrlich, who is regarded as the inventor of the concept of targeted therapy, received the Nobel Prize for Medicine. His initial perception leading to this theory was derived from observations that certain substances are capable of selectively staining either tissues or microorganisms. These observations culminated in the discovery of the inorganic mercury compound arsphenamine (Salvarsan) by Sahachiro Hata in the laboratory of Paul Ehrlich. Salvarsan might be regarded as the first effective "targeted" treatment for syphilis at that time. Tamoxifen (Nolvadex), an anti-estrogen, which was introduced in the early 1970s, was one of the first rationally designed targeted anti-tumour drugs. Since the 1970s a dramatic development of new molecular technologies occurred, culminating, for example, into the Human Genome Project and the public availability of various gene and protein databases, such as the Cancer Genome Anatomy Program established by the National Cancer Institute. Genomics, proteomics, structural genomics, transcriptomics, and high-throughput screening technologies for identification of targeted drugs are now available, which were almost unimaginable only a few years ago. Over 500 kinases are known of which about 250 have been cloned and are available to directly evaluate the activity of novel drug candidates. These technologies in conjunction with bio-informatic and chemical tools allow us to design novel molecules, and consequently tailor drug therapy to specific targets within tumours.

Speciation Analysis of Mercury in Cereals by Liquid Chromatography Chemical Vapor Generation Inductively Coupled Plasma-Mass Spectrometry

A simple and rapid procedure for the separation and determination of inorganic, methyl, and ethyl mercury compounds was described using liquid chromatography (LC) followed by vapor generation inductively coupled plasma-mass spectrometry (VG-ICP-MS). Well resolved chromatograms were obtained within 5 min by reversed-phase liquid chromatography with a C8 column as the stationary phase and a pH 4.7 solution containing 0.5% v/v 2-mercaptoethanol and 5% v/v methanol as the mobile phase. The separated mercury compounds were converted to mercury vapors by an in situ nebulizer/vapor generation system for their introduction into ICP. The concentrations of NaBH4 and HNO3 required for vapor generation were also optimized. The method was applied for the speciation of mercury in reference materials NIST SRM 1568a Rice Flour and NIST SRM 1567a Wheat Flour and also rice flour and wheat flour samples purchased locally. The accuracy of the procedure was verified by analyzing the certified reference material NRCC DOLT-3 Dogfish Liver for methyl mercury. Precision between sample replicates was better than 13% for all the determinations. The detection limits of the mercury compounds studied were in the range 0.003-0.006 ng Hg mL(-1) in the injected solutions, which correspond to 0.02-0.06 ng g(-1) in original flour samples. A microwave-assisted extraction procedure was adopted for the extraction of mercury compounds from rice flour, wheat flour, and fish samples using a mobile phase solution.

Sensitive Mercury Speciation by Reversed-Phase Column High-Performance Liquid Chromatography with UV-Visible Detection After Solid-Phase Extraction Using 6-Mercaptopurine and Dithizone

A highly selective and sensitive method was developed for preconcentration of inorganic and organic mercury compounds followed by reversed-phase column high-performance liquid chromatography (RP-HPLC) with UV-visible detection. The method was based on the reaction of mercury with 6-mercaptopurine and solid-phase extraction (SPE) of the complex on an octadecylsilane (C18) cartridge. The complex was then treated with ammoniacal dithizone solution, and the complexes of inorganic and organic mercury with dithizone were eluted by methanol. The speciation analysis of methylmercury (MeHg), phenylmercury (PhHg), and inorganic Hg (II) was carried out by RP-HPLC. Some experimental variables that influence the SPE and derivatization, such as pH, chelating and derivatizing agent concentration, and surfactant addition, were investigated. The calibration graphs of MeHg, PhHg, and Hg (II) were linear [correlation coefficient (r) > 0.999] from the detection limits (0.12, 0.16, and 0.14 ng) to 8.5, 6.0, and 6.7 ng Hg, respectively. By applying the SPE procedure, a 100-fold concentration of the sample was obtained. The procedure was applied to sea water and tuna fish samples. The method's accuracy was investigated by using tuna fish certified reference material BCR 464 and by spiking the samples with different amounts of MeHg, PhHg, and Hg (II). The average recoveries of MeHg, PhHg, and Hg (II) from spiked samples (0.1-2.0 microg/L Hg) were 96 +/- 4, 98 +/- 3, and 104 +/- 4%, respectively.

Soil Remediation – Mercury Speciation in Soil and Vapor Phase During Thermal Treatment

Spectroscopic (XRD, XPS, ICP-MS and AAS) and microscopic (ESEM) techniques have been used in order to study the chemical effects with emphasis on mercury speciation, during thermal treatment of a mercury contaminated soil. In the untreated soil, mercury was found concentrated in spherical particles, which were successively broken down upon thermal treatment. Hg0 and inorganic mercury compounds (presumably HgO(s) and HgSO4(s)) could be detected. No (CH3)2Hg and only traces of CH3Hg+ could be found. The dependence on temperature and heating time indicated that the evaporation of mercury from the soil was partly controlled by diffusion mechanisms. Mercury volatilized in two separate stages during heating; initial elemental vaporization, and subsequent volatilization of the oxide or sulfate phase at higher temperatures (>230°C). By thermal treatment at 470°C and 20 min, a removal of >99% of the mercury could be achieved.

Assessment of Chemcatcher passive sampler for the monitoring of inorganic mercury and organotin compounds in water

Several configurations of receiving disks and diffusion membranes were tested for monitoring mercury and organotin compounds (monobutyltin, dibutyltin, tributyltin, and triphenyltin) in water with a passive sampler. This passive sampler is based on the diffusion of these compounds through a specific diffusion-limiting membrane and their subsequent accumulation on a specific receiving phase, all materials being commercially available. The proposed sampler for inorganic mercury comprises a 47-mm Empore™ chelating disk as receiving phase and polyethersulfone as diffusion membrane. For monitoring organotins, the receiving phase is a 47-mm Empore™ C18 disk, and the diffusion membrane is cellulose acetate. ICP-MS and GC-ICPMS/GC-FPD were used for inorganic mercury and the organotins analysis, respectively. The effects of environmental variables such as pH and salinity that could influence accumulation of test substances in receiving phase were studied. Linear uptake for all compounds was observed for at least 14 days of exposure at a constant aqueous analyte concentration in a flow-through system under controlled conditions of temperature, turbulence, and analyte concentration. Compound-specific sampling rates at 11°C and simulated water turbulence of 40cms−1 varied between 0.018 and 0.137Ld−1. Compounds collected by the sampler exhibited detection limits ranging between 0.7 and 5.9ngL−1. The feasibility of using these samplers in the field was tested in a polluted commercial harbour. The behaviour of the samplers to monitor target compounds was compared with those obtained from spot samples of water taken throughout the field deployment period. Data from laboratory studies and field trial support the feasibility of these samplers to measure the freely dissolved fraction of these important target analytes in water.

Speciation analysis of mercury contaminants in water samples by RP-HPLC after solid phase extraction on modified C 18 extraction disks with 1,3-bis(2-cyanobenzene)triazene

A highly sensitive and accurate method for preconcentration and determination of ultra trace amounts of inorganic mercury and organomercury compounds in different water samples is proposed. The preconcentration is achieved using octadecyl silica (C 18) extraction disks modified with 1,3-bis(2-cyanobenzene)triazene (CBT). The retained analytes as their triazenide complexes on the solid phase was eluted with 10 ml acetonitrile and measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Type and amount of eluent, pH, amount of CBT, flow rates of sample solution and eluent have been optimized in order to obtain quantitative recovery of the analytes. The effect of interfering ions, such as Cu 2+, Mn 2+, Fe 2+, Al 3+, Zn 2+, Cd 2+, Ca 2+, Mg 2+, Ba 2+, Pb 2+, K + and Na + usually present in water samples on the recovery of the analytes has also been investigated. The enrichment factor of 100 was obtained for all mercury species and the analytical detection limits of phenylmercury, methylmercury and Hg 2+ were found as 0.8, 1.0 and 1.3 ng l − 1 , respectively. Stability of mercury species after extraction on the modified disks was studied and the results showed that complexes collected on the disks were stable for at least 5 days. The proposed method has been applied to the quantitative determination of mercury species in natural and synthetic water samples with recoveries more than 90%.

Genotoxicity and Cytotoxicity in Urothelial Cells of Women Exposed to Inorganic Mercury

ISEE-869 Objective: To investigate biomarkers of genotoxicity and cytotoxicity in urothelial cells of women exposed to inorganic mercury in a mining area, because mercury has been associated with genotoxic and cytotoxic effects in humans and animal models, and nevertheless mercury has not been considered carcinogenic in humans, even though genotoxic effects by exposure to inorganic mercury (Hg) compounds have been observed. Material and Methods: A cross sectional study was performed for evaluating exposure to Hg in a mining area (Hg in soil ranged between 50 to 100,000 ppb). The micronucleus assay in urothelial cells was performed in 115 women who were 12 to 84 years of age. Spot first morning urine samples were taken for determining Hg and creatinine as well as to isolate cells and to quantify cytogenetic damage. Results: The results show significant evidence of the possible association existing between genotoxicity and/or cytotoxicity and the presence of Hg in urine (HgUCr: geometric average was of 4.9 (rank 0.04–85 μg/g). In the multivariate model (generalized linear model) after controlling for other covariates, we observed that for each increment of 10 μg/g of HgUCr the odds ratio (OR) of developing some genotoxic effect increased by 2.37 times (IC 95% 1.79–2.84) while for cytotoxic damage in urothelial cells the OR was of 1.34 (IC 95% 1.10–1.79). Conclusions: In conclusion, living in a mining area with exposure to inorganic Hg and having mercury in urine, besides being exposed to other risk factors (ingestion of coffee, tobacco, and alcohol consumption) increase the possibilities of developing urothelial cytotoxicity and genotoxicity.

In Vitro Human Immune Response to Low Dose Mercury (89.11)

Abstract There is growing recognition that exposures to both inorganic and organic mercury (Hg) compounds can affect immune function, with potential consequences for host resistance to infections and risks of autoimmune disease. However, there is little information on human responses to low level Hg exposures. For this reason, we are investigating immune function using in vitro exposures to Hg in primary cultures of human peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. PBMCs were exposed to a concentration of HgCl2 up to 200 nM (which roughly corresponds to a whole blood concentration at the upper range of occupationally exposed or fish eating populations), in the presence and absence of activation by LPS. We found that these concentrations are non-cytotoxic and do not affect cell subsets or markers of cell activation. However, in PBMCs from both males and females, dose-dependent increases in both TNFα and IL-6 were observed in cell culture supernatants in the presence of LPS. No changes were observed in IFNγ, IL-4, IL-5, or IL-13 levels. No changes were seen in any cytokine in the absence of LPS. The results indicate that Hg can exert significant effects on signal transduction pathways in human cells in vitro, at exposures that are non-cytotoxic and environmentally relevant.