Monic acid A: a biomarker in clinical intra-nasal mupirocin medication for MRSA decolonisation.

CONTEXT: Mupirocin (BactrobanR) is widely prescribed for intra-nasal decolonisation of MRSA for in-patients awaiting surgery or self-medicated for out-patients although adherence for the latter is not monitored. Non-adherence is a widespread pharmaceutical problem but could encourage selection of antibiotic resistance. Mupirocin is only a topical antibiotic because it decomposes in stomach acidity to monic acid A, but this has not previously been exploited as a biomarker for clinical intra-nasal medication. MATERIALS AND METHODS: Urine from three catheterised patients in two London hospitals during and after mupirocin medication, was passed through Waters Oasis cartridges to isolate organic acids. Sensitive LC-MS-MS analysis for monic acid A in methanolic eluate has been developed to identify ∼10 pg. RESULTS: Monic acid A was quantified in all samples from one patient, translating into 6-46 ng from 12 mg mupirocin, assuming 1 L daily urine output. However, no urinary monic acid A was detected for two other patients. DISCUSSION AND CONCLUSIONS: Consistent occurrence of monic acid A in urine of one mupirocin patient shows for the first time that antibiotic distribution across nasal mucous membranes had generally been maintained during medication. In contrast, consistent absence in the two other patients requires wider study in hospital.

H NMR spectroscopy-based metabolomic assessment of uremic toxicity, with toxicological outcomes, in male rats following an acute, mid-life insult from ochratoxin a.

Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. (1)H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by (1)H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic.

HPLC/MS/MS methodology for sensitive quantitation of monic acid A, the metabolic product of the antibiotic mupirocin.

Patients who are treated by self-medication with intranasal mupiricin (Bactroban™) for controlling meticillin-resistant Staphylococcus aureus may, or may not, adhere to their regimen. Herein, we describe a potential methodology for assessing adherence by measuring the gastric degradation product, monic acid A (MA), as a biomarker in urine. MA was isolated (~80% recovery) through a Waters Oasis HLB cartridge and detected (e.g. 25 pg on the column) by HPLC/MS/MS (API4000). Within a calculated 10(6)-fold margin, this analytical sensitivity should facilitate urinary MA quantitation if, for example, 1% of intranasal mupirocin is swallowed and degraded characteristically to MA by gastric acidity.

Structures of covalent adducts between DNA and ochratoxin a: a new factor in debate about genotoxicity and human risk assessment.

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after (32)P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined (32)P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3'dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

Comments on "Ochratoxin A: In utero Exposure in Mice Induces Adducts in Testicular DNA. Toxins 2010, 2, 1428-1444"-Mis-Citation of Rat Literature to Justify a Hypothetical Role for Ochratoxin A in Testicular Cancer.

A manuscript in the journal recently cited experimental rat data from two manuscripts to support plausibility of a thesis that ochratoxin A might be a cause of human testicular cancer. I believe that there is no experimental evidence that ochratoxin A produces testicular cancer in rats or mice.

A pilot study of nuclear instability in archived renal and upper urinary tract tumours with putative ochratoxin aetiology.

DNA ploidy measurement has been applied uniquely to wax-embedded tissue of primary renal cell and metastatic tumours of a key experimental researcher on porcine ochratoxicosis, a control, and four transitional cell carcinomas from cases of Balkan endemic nephropathy. Primary renal tumour was diploid, and hyperdiploid metastasis was within the lower ploidy range for typical renal cell carcinoma. Three Balkan primary tumours showed extensive aneuploidy indicating marked nuclear instability, similar to model rat renal carcinoma caused by ochratoxin A. In contrast, much less nuclear instability in the putative occupational ochratoxicosis case fitted poorly with the ochratoxin A model.

Oncological outcomes in rats given nephrocarcinogenic exposure to dietary ochratoxin a, followed by the tumour promoter sodium barbital for life: a pilot study.

The potent experimental renal carcinogenesis of ochratoxin A (OTA) in male rats makes the dietary contaminant a potential factor in human oncology. We explored whether the tumour promoter sodium barbitate could shorten the otherwise long latency between exposure to toxin and tumourigenesis. Young rats, of a hybrid in which mononuclear leukaemia was rare, were given feed contaminated (5 ppm) with OTA for 36 weeks to initiate renal tumourigenesis. Some individuals were thereafter given sodium barbitate (500 ppm in drinking water) for life. Pathological outcomes were studied at or near the end of natural life. Renal tumours in males given barbitate became evident after latency of one year, but only slightly before those without barbitate. In contrast, female mammary tumourigenesis was advanced by at least 6 months synchronously in all rats given the OTA-barbitate regimen compared to tumourigenesis in controls. Diagnosis of malignant mammary angiosarcoma in a female given the OTA-barbitate regimen is a new finding in the rat. The long latency of OTA-induced renal tumourigenesis was not notably susceptible to accelerated promotion by barbitate, contrasting with an apparently marked effect of barbitate on development of mammary tumours.

Pathological outcomes in kidney and brain in male Fischer rats given dietary ochratoxin A, commencing at one year of age.

Malignant renal carcinoma, manifest in morbid ageing rats, is the striking component of an otherwise silent response after about nine months of exposure to ochratoxin A in the first year of life (daily intake ~100-250 µg/kg body weight). Reasons for the long latency are unclear, as is whether there would be a similar carcinogenic response if toxin exposure started at one year of age. Therefore, 24 male Fischer rats were given 100 µg ochratoxin A as a daily dietary contaminant for 35 weeks from age 50 weeks. Plasma ochratoxin A concentration reached a maximum value of ~8 µg/mL within one month of starting the toxin regimen. No renal carcinomas occurred. Four renal adenomas, two of which were only microscopic, were found among the six rats surviving for 110 weeks. The findings raise new questions about a difference between young adults and mature adults in sensitivity of male rats to the ochratoxin A-induced DNA damage necessary for renal carcinogenesis. A pilot histological study of perfuse-fixed brains of the toxin-treated rats showed no gross abnormalities, correlating with the consistent absence of behavioral or neurological disorders from chronic ochratoxin A exposure regimens in the range 100-250 µg/kg/day during the second half of life. Reasoned questioning concerning ochratoxin A as a neurotoxic mycotoxin is made.

Contrasting nephropathic responses to oral administration of extract of cultured Penicillium polonicum in rat and primate.

Liquid- or solid substrate-cultured Penicillium polonicum administered in feed to rats over several days evokes a histopathological response in kidney involving apoptosis and abnormal mitosis in proximal tubules. The amphoteric toxin is yet only partly characterized, but can be isolated from cultured sporulating biomass in a fraction that is soluble in water and ethanol, and exchangeable on either anion- or cation-exchange resins. After several weeks of treatment renal proximal tubule distortion became striking on account of karyocytomegaly, but even treatment for nearly two years remained asymptomatic. Extract from a batch of solid substrate fermentation of P. polonicum on shredded wheat was incorporated into feed for rats during four consecutive days, and also given as an aqueous solution by oral gavage to a vervet monkey daily for 10 days. Treatment was asymptomatic for both types of animal. Rat response was evident as the typical renal apoptosis and karyomegaly. In contrast there was no such response in the primate; and neither creatinine clearance nor any haematological characteristic or serum component concentration deviated from a control or from historical data for this primate. The contrast is discussed concerning other negative findings for P. polonicum in pigs and hamsters. Renal karyomegaly, as a common rat response to persistent exposure to ochratoxin A, is not known in humans suspected as being exposed to more than the usual trace amounts of dietary ochratoxin A. Therefore the present findings question assumptions that human response to ochratoxin A conforms to that in the rat.

Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in male Dark Agouti rats.

In rats fed dietary ochratoxin A (5 ppm for 3, 6 or 9 months) no renal tumours occurred throughout natural life of the group treated for 3 months, during which the ochratoxin dose was 3 times that in the high dose group of the NTP study. Bilateral renal carcinoma occurred in one rat in the 6 month group. Four rats treated for 9 months developed unilateral renal carcinoma. Overall latency between ceasing toxin exposure and discovering tumours was 35-97 weeks. Experimental verification of a 'no observable effect level' was made for feed containing 400 ppb, equivalent to approximately 7 microg ochratoxin A/day for Dark Agouti rats for up to 2 years, during which mean daily dose commenced at approximately 50 microg/kg, but later for adults was in the range 30-20 microg/kg. This data doubles the daily in vivo threshold dose from the NTP study ( approximately 15 microg/kg), and could influence human risk assessment. An at least 3 month threshold period for exposure to exceptionally high daily OTA intake (90 microg; 640-450 microg/kg) raises doubts over interpretation of experimental molecular data for OTA exposure at lower dose for up to 3 months in studies aimed at understanding carcinogenic mechanism.

The role of tryptophan as a biosynthetic precursor of indole-diterpenoid fungal metabolites: continuing a debate.

Studies in the 1980s and 1990s on the origin of the indole moiety in fungal indole-diterpenoids using (14)C-labelled tryptophan consistently showed autoradiographic evidence but gave low % incorporation of the probe. Recent studies on a member of the group (nodulisporic acid A), using more specific (13)C methodology, demonstrated a role of the tryptophan biosynthetic pathway but, in failing to show involvement of end-product, concluded that the indole was derived from indole-3-glycerol phosphate and suggested that the previous (14)C data arose via metabolic scrambling of label. In considering the protocol for the (13)C studies, there is concern that the fungal material was starved of an exogenous nitrogen source and thus could have degraded added labelled tryptophan. Consequently, synthesis of the serine necessary for anabolic formation of tryptophan may have been constrained. It is suggested that (13)C studies on appropriate fungi early in the idiophase of submerged or surface fermentation should be made before the biosynthesis of indole-diterpenoids can become clearer.

Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion.

Experiments designed to reveal aspects of delivery of circulating ochratoxin A (OTA) to kidneys showed maximum plasma concentration within 3h of acute gavage, but this persisted for 4 days before decline. During long-term daily administration in feed, plasma values stabilised, proportional to dose and, for Fischer males, immediately followed an 8-10 day half-life upon ceasing OTA intake. In mature adult males, plasma OTA accumulated during the month after commencing daily intake of 100 microg. By comparison, in Dark Agouti males, lower steady state plasma values occurred and there was much shorter plasma OTA half-life (2-3 days). In F1 hybrid rats (Sprague Dawley x Fischer) males ingesting 100 microg OTA daily the steady state plasma value stabilised as in Fischer males, but females on the same diet accumulated OTA in plasma to nearly twice the value for males after only 5 months' exposure. Although OTA causes renal tumours in Fischer and Dark Agouti male rats in spite of marked difference in OTA elimination rates presently shown, understanding comparison of re-uptake along nephrons in rat and man in vivo is vital in applying rat toxicological data to assessment of risk of dietary OTA to humans.

Biosynthesis of scorpinone, a 2-azaanthraquinone from Amorosia littoralis, a fungus from marine sediment.

The biogenetic origin of the carbon atoms in the 2-azaanthraquinone scorpinone ( 1), produced by the rare fungus Amorosia littoralis isolated from marine sediment, was explored through isotopic enrichment studies utilizing [2- (13)C]-acetate and [1,2- (13)C]-acetate. The labeling results reveal a heptaketide precursor is involved in the biosynthesis of 1, as has been found for the structurally related naphthoquinone dihydrofusarubin. The previously identified naphthoquinone herbarin ( 2) was also isolated and appears to bear the same biogenetic relationship to 1 as the fusarubins do to the fungal 2-azaanthraquinone bostrycoidins.

An experimental strategy towards optimising directed biosynthesis of communesin analogues by Penicillium marinum in submerged fermentation.

It was previously demonstrated that a fungus producing communesin alkaloids, subsequently identified as Penicillium marinum, could also accept 6-fluoro analogues of tryptophan or tryptamine to form mono-fluoro-communesin analogues in addition to communesins. A strategy to increase the relative yield of analogues by mutation to impair decarboxylation of tryptophan has been studied. Four mutants with much reduced activity of tryptophan decarboxylase, and other phenotypic change, were selected from 1500 colonies from spores that survived a 99% kill treatment with N-methyl N-nitro N-nitrosoguanidine. Tlc assessment of cell-associated products from standard submerged fermentations showed that one non-sporing mutant apparently produced little or no communesins, but productivity was restored when grown in a medium supplemented with glutamine. However, more sensitive mass spectrometric analysis detected both communesins A and B in mycelium grown on a rich, yeast extract-sucrose agar, showing that deletion of communesin biosynthesis was not absolute. It was concluded that mutagenesis had generally achieved its objective, but that new literature on a putative role of aurantioclavine in communesin biosynthesis presented an additional challenge to integrate the prenylation of tryptophan before its decarboxylation, which is a characteristic of ergot alkaloid biosynthesis.

A radiochemical technique with potential for revealing novel fungal metabolites according to expression of specific biosynthetic activities.

Fungal secondary metabolites are mostly derived from a few key intermediates in primary metabolism, such as acetate and some amino acids. Classical screens for novel fungal compounds of possible industrial interest have used chromatographic displays of extract components, as was the practice for plant natural products, followed by structural determination and pharmacological study. In contrast, modern robotic screens usually focus initially on specific bioassay applied to fermentation products and crude extracts. Both strategies are expensive in terms of human resources and/or in sophisticated equipment. A relatively inexpensive technique, exploiting biosynthetic principles through use of 14C-labelled probes to recognise particular structural features by autoradiography of tlc plates is described and discussed. Application to 80 isolates of filamentous fungi from Caribbean and European/Mediterranean environments enabled recognition of arrays of metabolites according to their biosynthetic origin, showing the potential of the technique in novel product discovery in unsophisticated laboratory facilities, as exemplified by reference to subsequent discovery of novel metabolites produced by Amorosia littoralis.

Binding of ochratoxin A to a urinary globulin: a new concept to account for gender difference in rat nephrocarcinogenic responses.

SDS-gradient mini-gel electrophoresis and immunoblotting of urine of rats given ochratoxin A (OTA), showed OTA binding to an alpha2u-globulin. Perceived potential internalised delivery of OTA to proximal tubule epithelia by the carrier, specific only to adult male rats and augmenting other uptake mechanisms, suggests that some experimental nephrotoxicological data may not be appropriate for human risk assessment. Reexamination of female rat renal tumour histopathology of the NTP high dose OTA study showed all carcinomas were solitary, unilateral, microscopic and clinically insignificant at the 2-year end-stage. The novel concept, when consolidated further from our archived material, may moderate current perceptions of the human risk of traces of dietary OTA.

DNA ploidy distribution in renal tumours induced in male rats by dietary ochratoxin A.

DNA ploidy distribution, measured in experimental renal tumours that occurred in twelve ageing male Fischer rats derived from carcinogenicity experiments on ochratoxin A (OTA) in response to chronic dietary exposure, was diploid in all renal adenomas and aneuploid in all carcinomas, correlating with their typical organised and disorganised histopathology, respectively. Aneuploidy was also detected in renal tissue in which karyomegaly, induced by OTA, was analogous to that caused by the fungus Penicillium polonicum. Thus, the experimental rat renal carcinoma could arise within an adenoma directly from certain persistent karyomegalic tubular epithelial cells long after their particular genetic damage has been caused during a protracted period of OTA insult.

Amorosia littoralis gen. sp. nov., a new genus and species name for the scorpinone and caffeine-producing hyphomycete from the littoral zone in The Bahamas.

The new generic and species name Amorosia littoralis gen. sp. nov. is introduced for the conidial dematiaceous hyphomycete isolated from the littoral zone in The Bahamas and reported in 2001 to produce the novel aza-anthraquinone scorpinone, and also caffeine. No satisfactory generic placement was found at the time, but subsequent morphological and molecular investigations reveal that a new generic name is required. The new genus has some similarity to several fungi described in Trichocladium, but differs substantially from the type species of that genus in the form of the conidia and the lack of ornamentation. BLAST studies using the 18S and 28S rDNA gene sequences place the new genus in the Sporormiaceae. In addition to the morphological studies, an ultrastructural examination of the conspicuous porate septa of hyphae showed that they do not belong to a basidiomycete.

Natural and directed biosynthesis of communesin alkaloids.

A role for tryptophan, acetate, mevalonate and methionine in the biosynthesis of communesins A and B, novel structurally-related and biologically-active Penicillium metabolites, has been established by isotopic labelling techniques. The incorporation of (14)C-tryptamine has also been demonstrated. dl-2-(13)C-tryptophan specifically enriched two carbon atoms in the (13)C NMR spectrum, thereby defining the intra-molecular arrangement of the two tryptophan-derived moieties. Feeding differentially labelled precursors during communesin production showed that tryptophan and methionine are involved early in the biosynthesis and that mevalonate provides an isoprene which is added later. A biosynthetic pathway involving an early precursor based on tryptophan is proposed. Indole-N-((13)C-methyl) tryptophan was not incorporated into communesins implying that N-methylation of tryptophan is not the first step of the communesin biosynthetic pathway. During deamination of indole-N-((13)C-methyl) tryptophan to 1-(13)C-methylindole-3-carboxylic acid communesin biosynthesis was inhibited. Of several halogenated indoles tested for directed biosynthesis, only dl-6-fluoro-tryptophan and 6-fluoro-tryptamine caused accumulation of the corresponding monofluoro-analogues of communesins A and B.

Deuterium NMR used to indicate a common mechanism for the biosynthesis of ricinoleic acid by Ricinus communis and Claviceps purpurea.

Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct (2)H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative (2)H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.