Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars.

BACKGROUND: A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. METHODS: Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). RESULTS: CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ±â€¯0.02 vs 0.12 ±â€¯0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. CONCLUSIONS: We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.

SMT19969 as a treatment for Clostridium difficile infection: an assessment of antimicrobial activity using conventional susceptibility testing and an in vitro gut model.

OBJECTIVES: We investigated the efficacy of the novel antimicrobial agent SMT19969 in treating simulated Clostridium difficile infection using an in vitro human gut model. METHODS: Concentrations of the predominant cultivable members of the indigenous gut microfloras and C. difficile (total and spore counts) were determined by viable counting. Cytotoxin titres were determined using cell cytotoxicity and expressed as log10 relative units (RU). Clindamycin was used to induce simulated C. difficile PCR ribotype 027 infection. Once high-level cytotoxin titres (≥ 4 RU) were observed, SMT19969 was instilled for 7 days. Two SMT19969 dosing regimens (31.25 and 62.5 mg/L four times daily) were evaluated simultaneously in separate experiments. MICs of SMT19969 were determined against 30 genotypically distinct C. difficile ribotypes. RESULTS: SMT19969 was 7- and 17-fold more active against C. difficile than metronidazole and vancomycin, respectively, against a panel of genotypically distinct isolates (P < 0.05). Both SMT19969 dosing regimens demonstrated little antimicrobial activity against indigenous gut microflora groups except clostridia. SMT19969 inhibited C. difficile growth and repressed C. difficile cytotoxin titres in the gut model. CONCLUSIONS: These data suggest that SMT19969 is a narrow-spectrum and potent antimicrobial agent against C. difficile. Additional studies evaluating SMT19969 in other models of C. difficile infection are warranted, with human studies to place these gut model observations in context.

In vitro activity of cadazolid against clinically relevant Clostridium difficile isolates and in an in vitro gut model of C. difficile infection.

OBJECTIVES: We investigated the in vitro activity of cadazolid against 100 Clostridium difficile isolates and its efficacy in a simulated human gut model of C. difficile infection (CDI). METHODS: MICs of cadazolid, metronidazole, vancomycin, moxifloxacin and linezolid were determined using agar incorporation for 100 C. difficile isolates, including 30 epidemic strains (ribotypes 027, 106 and 001) with reduced metronidazole susceptibility, 2 linezolid-resistant isolates and 2 moxifloxacin-resistant isolates. We evaluated the efficacy of two cadazolid dosing regimens (250 versus 750 mg/L twice daily for 7 days) to treat simulated CDI. Microflora populations, C. difficile total viable counts and spores, cytotoxin titres, possible emergence of cadazolid, linezolid or quinolone resistance, and antimicrobial concentrations were monitored throughout. RESULTS: Cadazolid was active against all (including linezolid- and moxifloxacin-resistant) C. difficile strains (MIC90 0.125, range 0.03-0.25 mg/L). The cadazolid geometric mean MIC was 152-fold, 16-fold, 9-fold and 7-fold lower than those of moxifloxacin, linezolid, metronidazole and vancomycin, respectively. Both cadazolid dosing regimens rapidly reduced C. difficile viable counts and cytotoxin with no evidence of recurrence. Cadazolid levels persisted at 50-100-fold supra-MIC for 14 days post-dosing. Cadazolid inhibition of enumerated gut microflora was limited, with the exception of bifidobacteria; Bacteroides fragilis group and Lactobacillus spp. counts were unaffected. There was no evidence for selection of strains resistant to cadazolid, quinolones or linezolid. CONCLUSIONS: Cadazolid activity was greater than other tested antimicrobials against 100 C. difficile strains. Cadazolid effectively treated simulated CDI in a gut model, with limited impact on the enumerated gut microflora and no signs of recurrence or emergence of resistance within the experimental timeframe.

Successful treatment of simulated Clostridium difficile infection in a human gut model by fidaxomicin first line and after vancomycin or metronidazole failure.

OBJECTIVES: Fidaxomicin reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. We investigated fidaxomicin primary or secondary treatment efficacy using a gut model. METHODS: Four triple-stage chemostat gut models were inoculated with faeces. After clindamycin induction of CDI, fidaxomicin (200 mg/L twice daily), vancomycin (125 mg/L four times daily) or metronidazole (9.3 mg/L three times daily) was administered for 7 days. Following failure/CDI recurrence, fidaxomicin (200 mg/L twice daily, 7 days) was instilled. C. difficile (CD) total viable counts (TVC), spore counts (SP), toxin titres (CYT), gut bacteria counts and antimicrobial concentrations were measured throughout. RESULTS: Fidaxomicin instillation reduced CD TVC/SP and CYT below the limit of detection (LOD) after 2 and 4 days, respectively, with no CDI recurrence. Metronidazole instillation failed to decrease CD TVC or CYT. Vancomycin instillation reduced CD TVC and CYT to LOD by day 4, but SP persisted. Recurrence occurred 13 days after vancomycin instillation; subsequent fidaxomicin instillation reduced CD TVC/SP/CYT below the LOD from day 2. CD was isolated sporadically, with no evidence of spore recrudescence or toxin production. Fidaxomicin had a minimal effect on the microflora, except for bifidobacteria. Fidaxomicin was detected for at least 21 days post-instillation, whereas other antimicrobials were undetectable beyond ∼4 days. CONCLUSIONS: Fidaxomicin successfully treated simulated primary and recurrent CDI. Fidaxomicin was superior to metronidazole in reducing CD TVC and SP, and superior to vancomycin in reducing SP without recurrence of vegetative cell growth. Fidaxomicin, but not vancomycin or metronidazole, persisted in the gut model for >20 days after instillation.

Pharmacological characterization of 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a high-affinity antagonist selective for κ-opioid receptors.

2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human µ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 µM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.

Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum.

AIMS: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. METHODS AND RESULTS: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0.5%, 1%, 1.5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose-dependent fashion, whereas butyric acid production showed a significant linear increase. CONCLUSIONS: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro-organisms selected for the study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.

echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases.

BACKGROUND: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. RESULTS: We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. CONCLUSION: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye.

Use of the infant transwarmer mattress as an external warming modality in resuscitation from hypothermia.

Normothermia must be established in drowning victims before death may be declared, as the myocardium may remain resistant to stimulation at subnormal temperatures, and complete neurological recovery from submersion associated hypothermia has been reported. A safe and effective method of external re-warming is described that may prove particularly useful in the paediatric population.

Lesions in the central nucleus of the amygdala: discriminative avoidance learning, discriminative approach learning, and cingulothalamic training-induced neuronal activity.

The amygdala is critically involved in discriminative avoidance learning. Large lesions of the amygdala block discriminative avoidance learning and abolish cingulothalamic training-induced neuronal activity. These results indicated that amygdalar processing is critical for cingulothalamic plasticity. The larger lesions did not allow differentiation of the specific functioning of various amygdalar nuclei. Anatomical analysis showed that damage in the central (CE) nucleus of the amygdala was correlated with the severity of the behavioral deficit. The present study was carried out to determine whether smaller lesions, centered in the CE nucleus, would impair discriminative avoidance learning and block cingulothalamic plasticity. In addition, the possible role of the CE nucleus in appetitively motivated discriminative approach learning was examined for the first time. New Zealand White rabbits with CE nuclear lesions were first trained in the discriminative approach task. After attaining asymptotic performance, discriminative avoidance training sessions were alternated with continuing approach training sessions, one session each day. The rabbits with lesions were severely impaired in avoidance learning but showed no impairment of approach learning. Surprisingly, the attenuating effects of the lesions on cingulothalamic training-induced neuronal activity were more prevalent during approach learning than during avoidance learning. These results indicated that avoidance learning can be impaired by lesions centered in the CE nucleus that leave cingulothalamic plasticity largely intact and that the CE nucleus is involved in extra-cingulothalamic learning processes.

Studies directed at the use of a parallel synthesis matrix to increase throughput in an in vivo assay.

Heparin is the anticoagulant of choice for hospitalized patients, but it is dosed only by injection because it is not absorbed following oral administration. We have discovered and prepared compounds (delivery agents) that facilitate the gastrointestinal absorption of heparin in rats, monkeys, and humans when given orally. We are currently developing a parallel synthesis approach to increase our delivery agent screening throughput in vivo. This approach has been used to produce micromolar quantities of compounds for testing in rats in a 5 x 5 parallel synthesis array. Using an amine benzoylation reaction sequence, 10 mixtures were prepared. These mixtures contained equal weight quantities of five N-substituted, non-alpha, amino acid delivery agents. Each of these mixtures was orally administered to rats in combination with heparin, and plasma clotting times (APTT) were measured to determine activity. Deconvolution of the data accurately identified the most active individual components. Independent synthesis of these compounds verified their activity. This parallel synthesis approach is an effective tool for the screening of oral heparin delivery agents and has increased screening throughput significantly.

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation.

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.

Using treatment failure under effective directly observed short-course chemotherapy programs to identify patients with multidrug-resistant tuberculosis.

SETTING: Public ambulatory care centers in three districts of northern metropolitan Lima, Peru. OBJECTIVE: To document drug resistance patterns of isolates of Mycobacterium tuberculosis from patients identified as treatment failures under a model tuberculosis (TB) control program based on directly observed, short-course chemotherapy (DOT-SCC). DESIGN: Case series. RESULTS: In a referred, consecutive sample of 173 patients identified as treatment failures on DOT-SCC, 160 (92.5%) had culture-positive TB. Of those 160, 150 (93.8%) had active, pulmonary multidrug-resistant TB (MDR-TB, resistance to at least isoniazid [INH] and rifampicin [RIF]). Sixty of the 150 (40.0%) had isolates resistant to at least INH, RIF, ethambutol (EMB) and pyrazinamide (PZA), the initial first-line empiric treatment regimen used locally. Forty-four (29.3%) had isolates resistant to at least INH, RIF, EMB, PZA and streptomycin (SM), the first retreatment regimen. This series of patients had isolates resistant to a mean of 4.5 of the ten drugs tested. The local profile of multidrug resistance is very different from that obtained from national data from Peru. CONCLUSION: In this setting, treatment failure on DOT-SCC is strongly predictive of active MDR-TB. Because of existing local drug resistance patterns in northern Lima, 89.3% of MDR-TB patients identified as treatment failures will receive ineffective therapy with two or fewer secondary TB drugs if they are given the five-drug empiric retreatment regimen endorsed by the World Health Organization. Further short-course chemotherapy for these patients would only serve to amplify ominous existing drug resistance patterns.

Developmental changes in eye-blink conditioning and neuronal activity in the cerebellar interpositus nucleus.

Neuronal activity was recorded in the cerebellar interpositus nucleus in infant rats during classical conditioning of the eye-blink response. The percentage and amplitude of eye-blink conditioned responses increased as a function of postnatal age. Learning-specific neuronal activity in the cerebellum emerged ontogenetically in parallel with the eye-blink conditioned response. There were also age-specific changes in neuronal activity after the onset of the conditioned and unconditioned stimuli. The results indicate that the development of the eye-blink conditioned response may depend on the development of stimulus-evoked neuronal responses and learning-specific plasticity in the cerebellum. Functional immaturity in the afferent neural pathways may limit the induction of neural plasticity in the cerebellum and thereby limit the development of the eye-blink conditioned response.

The role of dermal irritation in the skin tumor promoting activity of petroleum middle distillates.

Petroleum middle distillates (PMDs), a class of hydrocarbons which boil between 350-700 degrees F, are tumor promoters in mouse skin. The promotional activity is produced under conditions that also result in local changes, including chronic irritation and epidermal hyperplasia. The present study was conducted by comparing equal weekly doses of irritating and minimally or nonirritating test materials, to assess whether tumor promotion was a secondary response to these effects. Four PMDs, C10-C14 normal paraffins (NP), lightly refined paraffinic oil (LRPO), Jet Fuel A (JF), and steam-cracked gas oil (SCGO), were evaluated. Test materials were applied undiluted (2x/week) or as 28.6% (7x/week) or 50% (4x/week) concentrations in mineral oil for 52 weeks following initiation with dimethylbenzanthracene (DMBA). When applied undiluted, all materials produced moderate irritation and significant increase in tumor incidence. When NP, LRPO, or JF were applied in mineral oil diluent, skin irritation was generally ameliorated and few, if any, tumors were produced. SCGO was irritating and produced a significant increase in tumor frequency when administered in mineral-oil diluent. These data indicate that the promotional activity of straight-run PMDs is likely related to chronic irritation at the application site and not to dose. Thus, when used appropriately in the absence of prolonged irritation, these materials should not present a tumorigenic hazard to humans.

Intergenic splicing between a HERV-H endogenous retrovirus and two adjacent human genes.

We previously reported that a long terminal repeat (LTR) of a human endogenous retrovirus of the HERV-H family promotes expression of a cellular fusion transcript in teratocarcinoma cell lines. This transcript was termed PLA2L due to two regions of similarity to the secreted form of phospholipase A2. In this study, evidence is presented indicating that this transcript appears to be the result of intergenic splicing between the HERV-H element and two independent downstream genes. The 5' gene has been named HHLA1 (HERV-H LTR-associating 1) and is of unknown function but shows sequence conservation in other mammals. The 3' gene is now known to encode human otoconin-90 (OC90) which, in mice, is a major protein expressed in the fetal inner ear. Evidence for intergenic splicing of these two genes includes: (1) the isolation of LTR-driven HHLA1 transcripts, unspliced to otoconin-90 exons, with variable sites of polyadenylation; (2) the cloning of both the putative human intergenic genomic region and the novel 5' terminus of the mouse otoconin-90 gene; (3) the identification of homologous potential signal sequences in the 5' region of mouse otoconin-90 and in the middle of the PLA2L transcript; and (4) the lack of detectable chromosomal rearrangements involving this region in teratocarcinoma cells. The PLA2L transcript therefore represents a rare example of intergenic splicing of two closely linked genes. We hypothesize that human HHLA1 and OC90 are normally expressed independently from different promoters but are expressed from the LTR promoter and spliced together in teratocarcinoma cells. It is tempting to speculate that the high activity of the LTR promoter in this cell type may induce transcriptional fusion between these two genes.

Impairment of cingulothalamic learning-related neuronal coding in rabbits exposed to cocaine in utero: general and sex-specific effects.

Neuronal activity was recorded in the cingulate cortex and the limbic thalamus in Dutch-belted rabbits (Oryctolagus cuniculus) exposed to cocaine (8 mg/kg/day i.v.) or saline in utero during acquisition and reversal learning of a discriminative avoidance response. Anterior cingulate cortical excitatory training-induced activity (TIA) was attenuated in cocaine-exposed female rabbits during acquisition and reversal learning, but only during reversal learning in male rabbits. Posterior cingulate cortical excitatory TIA was lessened in cocaine-exposed rabbits during acquisition, whereas discrimination between the positive and negative cues was enhanced. Neuronal firing was attenuated in the anterior ventral thalamus in cocaine-exposed rabbits during acquisition and reversal learning. Behavioral learning was normal in cocaine-exposed rabbits. Other data suggest that rabbits exposed to cocaine in utero exhibit a learning deficit when trained with nonsalient cues.

Assessment of the utility of the micronucleus test for petroleum-derived materials.

The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil (CCCO). This material contains high levels of polycyclic aromatic constituents (PAC) and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay.

Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates.

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that this effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-07); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia. PMD were administered (2 x/week for 2 weeks) to skin of CD-1 mice. Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but > 1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e., API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1 (v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.