Prostaglandin release from macrophages: An assay system for anti-inflammatory drugs in vitro

It is the intention of this communication to present a simple and reliable in vitro system for the evaluation of anti-inflammatory drugs. The system described appears to combine the advantages of two established procedures, i.e. measurement of the inhibition of prostaglandin (PG) synthesis in vitro using microsomal enzyme preparations and the measurement of the inhibitory effects of drugs on PG synthesis in vivo avoiding major limitations. We propose to measure the effect of drugs on the PG release from mouse peritoneal macrophages in vitro. The influence of culture conditions, phagocytosis and drugs on the PG release from these cells is described. We found that antibody-coated erythrocytes are especially suitable triggers of PG release, and that the release can be inhibited by steroidal and nonsteroidal anti-inflammatory drugs in concentrations resembling their effective doses in vivo.Dedicated to K. Bucher on the occasion of his 65th birthday.     

DRD2 -141C insertion/deletion polymorphism is not associated with schizophrenia: results of a meta-analysis.

The gene DRD2, which codes for dopamine receptor D2, has been considered a prime candidate for allelic association testing with schizophrenia based on the strong evidence for involvement of this protein in disease pathophysiology. Recent meta-analyses confirmed a small but reliable association between schizophrenia and the cysteine-coding allele of the Cys311Ser polymorphism of DRD2. In the present study, we sought to determine if another polymorphism (the -141C insertion/deletion) in the same gene, which has been reported to be associated with schizophrenia in several individual studies, would show a similar pattern of association with the disease in a pooled dataset. The pooled odds ratio for the insertion allele obtained from 10 case-control studies was 1.1, which was not significant (P = 0.580); however, there was marked heterogeneity among the findings of individual studies, suggesting that some underlying factor influenced the size of their observed effects. Yet, neither ethnicity, the age of the control group, nor the gender composition of the samples reliably influenced effect size. Because linkage disequilibrium patterns between various DRD2 polymorphisms are not yet known, it remains possible that divergent meta-analytic findings at both commonly examined mutation sites within DRD2 are accurate. Haplotype analysis within this gene would be useful for definitively specifying the role of this gene in the etiology of schizophrenia.     

Efficient oxidation of promutagenic hydroxymethylpyrenes by cDNA-expressed human alcohol dehydrogenase ADH2 and its inhibition by various agents

Alkylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulphation to electrophilically reactive esters. However, we previously found that the predominant biotransformation route for the hepatocarcinogen 1-hydroxymethylpyrene (1-HMP) in the rat in vivo is the oxidation of the side chain by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases to the carboxylic acid. Inhibition of this pathway by ethanol (competing ADH substrate) or 4-methylpyrazole (ADH inhibitor) led to a dramatic increase in the 1-HMP-induced DNA adduct formation in rat tissues in the preceding study. In order to elucidate the role of individual ADHs in the metabolism of alkylated polycyclic aromatic hydrocarbons, we expressed the various members of the human ADH family in bacteria. Cytosolic preparations from bacteria expressing ADH2 clearly oxidized hydroxymethylpyrene isomers (1-, 2- and 4-HMP) with the highest rate. This form was purified to near homogeneity to perform detailed kinetic analyses. High catalytic efficiencies (V(max)/K(m)) were observed with HMPs. Thus, this value was 10,000-fold higher for 2-HMP than for the reference substrate, ethanol. The corresponding aldehydes were also efficiently reduced by ADH2. 4-Methylpyrazole inhibited the oxidation of the HMP isomers as well as the reverse reaction. Daidzein, cimetidine and the competing substrate ethanol were further compounds that inhibited the ADH2-mediated oxidative detoxification of 1-HMP.     

Oxidation of alcohols and reduction of aldehydes derived from methyl- and dimethylpyrenes by cDNA-expressed human alcohol dehydrogenases

Some methylated pyrenes can form DNA adducts in rat tissues after benzylic hydroxylation and sulpho conjugation. However, oxidation of the intermediate alcohols to carboxylic acids is an important competing pathway leading to detoxification. We previously showed that co-administration of ethanol or 4-methylpyrazole strongly enhances DNA adduct formation by 1-hydroxymethylpyrene, indicating an involvement of alcohol dehydrogenases (ADHs) in the detoxification. This mechanism may be involved in the observed synergism of smoking and alcohol consumption in certain human cancers. In a preceding study, cDNA-expressed human ADH2 efficiently oxidised 1-, 2- and 4-hydroxymethylpyrene; these reactions were inhibited in the presence of ethanol or 4-methylpyrazole. Here we report that ADH1C, ADH3 and ADH4 also show substantial activity towards these substrates and two further congeners, 1-hydroxymethyl-6-methylpyrene and 1-hydroxymethyl-8-methylpyrene. All four ADH forms also catalysed the reverse reaction, implying that the aldehydes have to be sequestered by other enzymes, such as aldehyde dehydrogenases, for final detoxification. ADH1C and ADH4 activities towards hydroxymethylpyrenes were more strongly inhibited in the presence of ethanol and 4-methylpyrazole than those of ADH2. ADH3 was only inhibited at very high concentrations of the modulators. In conclusions, several human ADHs are capable of detoxifying benzylic alcohols of alkylated polycyclic aromatic hydrocarbons. However, some competing substrates and inhibitors may affect all these redundant detoxification systems, although to various extents.     

Bioactivation of food genotoxicants 5-hydroxymethylfurfural and furfuryl alcohol by sulfotransferases from human,mouse and rat: a comparative study

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are moderately potent rodent carcinogens that are present in thermally processed foodstuffs. The carcinogenic effects were hypothesized to originate from sulfotransferase (SULT)-mediated bioactivation yielding DNA-reactive and mutagenic sulfate esters, a confirmed metabolic pathway of HMF and FFA in mice. It is known that orthologous SULT forms substantially differ in substrate specificity and tissue distribution. This could influence HMF- and FFA-induced carcinogenic effects. Here, we studied HMF and FFA sulfoconjugation by 30 individual SULT forms of humans, mice and rats. The catalytic efficiencies (k _cat/K _M) of HMF sulfoconjugation of human SULT1A1 (13.7 s^?1 M^?1), mouse Sult1a1 (15.8 s^?1 M^?1) and 1d1 (4.8 s^?1 M^?1) and rat Sult1a1 (5.3 s^?1 M^?1) were considerably higher than those of all other SULT forms investigated (≤0.73 s^?1 M^?1). FFA sulfoconjugation was monitored using adenosine as a nucleophilic scavenger for the reactive 2-sulfoxymethylfuran (t _1/2 = 20 s at 37 °C). The resulting adduct N ⁶-((furan-2-yl)methyl)-adenosine (N ⁶-MF-A) was quantified by isotope-dilution UPLC-MS/MS. The rates of N ⁶-MF-A formation showed that hSULT1A1 and its orthologues in mice and rats were also the most important contributors to FFA sulfoconjugation in each of the species. Taken together, the catalytic capacity of hSULT1A1 is comparable to that of mSult1a1 in mice, the species in which carcinogenic effects of HMF and FFA were detected. This is of primary concern due to the expression of hSULT1A1 in many different tissues.     

Rapid and Successful Implementation of a COVID-19 Convalescent Plasma Programme—The South African Experience

Background: COVID-19 convalescent plasma (CCP) has been considered internationally as a treatment option for COVID-19. CCP refers to plasma collected from donors who have recovered from and made antibodies to SARS-CoV-2. To date, convalescent plasma has not been collected in South Africa. As other investigational therapies and vaccination were not widely accessible, there was an urgent need to implement a CCP manufacture programme to service South Africans. Methods: The South African National Blood Service and the Western Cape Blood Service implemented a CCP programme that included CCP collection, processing, testing and storage. CCP units were tested for SARS-CoV-2 Spike ELISA and neutralising antibodies and routine blood transfusion parameters. CCP units from previously pregnant females were tested for anti-HLA and anti-HNA antibodies. Results: A total of 987 CCP units were collected from 243 donors, with a median of three donations per donor. Half of the CCP units had neutralising antibody titres of >1:160. One CCP unit was positive on the TPHA serology. All CCP units tested for anti-HLA antibodies were positive. Conclusion: Within three months of the first COVID-19 diagnosis in South Africa, a fully operational CCP programme was set up across South Africa. The infrastructure and skills implemented will likely benefit South Africans in this and future pandemics.     

Metabolic activation of furfuryl alcohol: formation of 2-methylfuranyl DNA adducts in Salmonella typhimurium strains expressing human sulfotransferase 1A1 and in FVB/N mice

Furfuryl alcohol, formed by acid- and heat-induced dehydration from pentoses, is found in many foodstuffs. It induced renal tubule neoplasms in male B6C3F1 mice and nasal neoplasms in male F344/N rats in a study of the National Toxicology Program (NTP). However, furfuryl alcohol was negative in the standard Ames test and in a battery of in vivo mutagenicity tests. Here, we show that furfuryl alcohol is mutagenic in Salmonella typhimurium TA100 engineered for expression of human sulfotransferase (SULT) 1A1. This finding suggests that furfuryl alcohol is converted by intracellular sulfo conjugation to 2-sulfo-oxymethylfuran, an electrophile reacting with DNA. We detected nucleoside adducts of 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxycytidine in porcine liver DNA incubated with freshly prepared 2-sulfo-oxymethylfuran. The main adducts, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MFdG) and N(6)-((furan-2-yl)methyl)-2'-deoxyadenosine (N(6)-MFdA) were synthesized. Their structures were verified by NMR and mass spectrometry. Liquid chromatography-tandem mass spectrometry methods for the quantification of both adducts were devised. N(2)-MFdG and N(6)-MFdA were detected in DNA of furfuryl alcohol-exposed S.typhimurium TA100 expressing SULT1A1 and in DNA of liver, lung and kidney of FVB/N mice that had received ～390 mg furfuryl alcohol/kg body wt/day via the drinking water for 28 days. In summary, furfuryl alcohol is converted by sulfo conjugation to a mutagen. The detection of N(2)-MFdG and N(6)-MFdA in renal DNA of furfuryl alcohol-treated mice suggests that the neoplasms observed in this tissue in the study of the NTP may have been induced by 2-sulfo-oxymethylfuran.     

Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Inter- and intraclass correlations for three standard foot radiographic measurements for plantar surface angles. Which measure is most reliable?

BackgroundThe purpose of this study was to evaluate the reliability and reproducibility of three commonly used radiographic measures for plantar surface angles.MethodsThe calcaneal angle (CA), calcaneal pitch angle (CPA), and length-height index (LHI) was measured by three independent examiners on two occasions on lateral foot radiographs. Intra- and inter-rater correlations were calculated using a general linear estimate model and post-hoc tests for repeated measures. Bland–Altman’s plots with limits of agreement were used for observer differences in scores.ResultsThe intra-class correlations for the CA ranged from 0.91 to 0.94, for the CPA from 0.93 to 0.98, and for the LHI from 0.96 to 0.97. The inter-class correlations were 0.80 for CA, 0.83 for CPA and 0.93 for LHI.ConclusionsThe results of this study strongly suggest that the length-height index was the most consistent and reliable measure for arch height.Level of evidence: Diagnostic Level II, validity.