A Canadian Study of Cisplatin Metabolomics and Nephrotoxicity (ACCENT): A Clinical Research Protocol

Background: Cisplatin, a chemotherapy used to treat solid tumors, causes acute kidney injury (AKI), a known risk factor for chronic kidney disease and mortality. AKI diagnosis relies on biomarkers which are only measurable after kidney damage has occurred and functional impairment is apparent; this prevents timely AKI diagnosis and treatment. Metabolomics seeks to identify metabolite patterns involved in cell tissue metabolism related to disease or patient factors. The A Canadian study of Cisplatin mEtabolomics and NephroToxicity (ACCENT) team was established to harness the power of metabolomics to identify novel biomarkers that predict risk and discriminate for presence of cisplatin nephrotoxicity, so that early intervention strategies to mitigate onset and severity of AKI can be implemented. Objective: Describe the design and methods of the ACCENT study which aims to identify and validate metabolomic profiles in urine and serum associated with risk for cisplatin-mediated nephrotoxicity in children and adults. Design: Observational prospective cohort study. Setting: Six Canadian oncology centers (3 pediatric, 1 adult and 2 both). Patients: Three hundred adults and 300 children planned to receive cisplatin therapy. Measurements: During two cisplatin infusion cycles, serum and urine will be measured for creatinine and electrolytes to ascertain AKI. Many patient and disease variables will be collected prospectively at baseline and throughout therapy. Metabolomic analyses of serum and urine will be done using mass spectrometry. An untargeted metabolomics approach will be used to analyze serum and urine samples before and after cisplatin infusions to identify candidate biomarkers of cisplatin AKI. Candidate metabolites will be validated using an independent cohort. Methods: Patients will be recruited before their first cycle of cisplatin. Blood and urine will be collected at specified time points before and after cisplatin during the first infusion and an infusion later during cancer treatment. The primary outcome is AKI, defined using a traditional serum creatinine-based definition and an electrolyte abnormality-based definition. Chart review 3 months after cisplatin therapy end will be conducted to document kidney health and survival. Limitations: It may not be possible to adjust for all measured and unmeasured confounders when evaluating prediction of AKI using metabolite profiles. Collection of data across multiple sites will be a challenge. Conclusions: ACCENT is the largest study of children and adults treated with cisplatin and aims to reimagine the current model for AKI diagnoses using metabolomics. The identification of biomarkers predicting and detecting AKI in children and adults treated with cisplatin can greatly inform future clinical investigations and practices

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested

Development and application of models of intra-oral halitosis

Halitosis is a common condition with a multitude of oral and systemic factors implicated in its causality. Although, the pathological factors involved in the systemic causes of halitosis are varied, the oral factors are more consistent and mostly involve the production of malodorous volatiles by the oral microbiota. This phenomenon can therefore be modelled 'in vitro' and two such models, utilising planktonic oral bacteria and tongue-derived biofilms, were used to ascertain the effects of amino acids and foods in the generation of malodour, mainly focusing on the volatile, sulfur-containing compounds. To that end, simple amino acids as well as garlic, onion and coffee were evaluated in both the models. It was found that odorous food components could be retained and released within the biofilm model and that amino acids could be metabolised to a variety of different malodorous compounds. These models were applied in the evaluation of zinc, an active incorporated in oral healthcare products to combat malodour. Further, a thorough evaluation of the role of the bacterium, 'Solobacterium moorei' was undertaken, finding that the organism could generate copious amounts of hydrogen sulfide

'In vitro' growth characteristics and volatile sulfur compound production of 'Solobacterium moorei'

Solobacterium moorei has recently been implicated as a causative agent of halitosis. In vitro experiments to evaluate the role of S. moorei in halitosis have, however, been complicated by a paucity of information on the ideal conditions for culturing this organism. This work aimed to optimize a liquid culture medium for S. moorei, and to determine the growth-curve of the organism. Further, the ability of S. moorei to generate volatile sulfur compounds was investigated and compared quantitatively to other oral anaerobes by an optimized head-space gas chromatography method. Serum-supplementation of standard liquid growth media gave greater growth of S. moorei than non-supplemented broths, with the best medium found to be serum-supplemented tryptone soya broth. S. moorei was able to metabolize cysteine directly to hydrogen sulfide, but was unable to produce methanethiol from methionine. S. moorei produced 2-3 times more hydrogen sulfide (normalized for colony forming units) than Porphyromonas gingivalis and Veillonella dispar, but considerably less than Fusobacterium nucleatum. The study has identified reliable growth conditions for culture of S. moorei, which were employed to show that S. moorei has the requisite biochemistry consistent with a potential role in halitosis

Primer pairs complementary to the bacterial 16S gene used in quantifying bacterial biofilm populations in this study and the primer pairs used for mutagenesis.

<p>Primer pairs complementary to the bacterial 16S gene used in quantifying bacterial biofilm populations in this study and the primer pairs used for mutagenesis.</p

The relative mRNA expression of the stimulated cells and unstimulated controls at the 24h time point.

<p>Whiskers and outliers determined by the Tukey method. C = Control; Δ = PG343 biofilms; WT = W50 biofilms.</p

Cytokines that showed significant differences between the PG343 and W50 biofilm stimulations at 4h and 24h.

<p>PG343 = Δ; W50 = WT; C = Controls. Median is denoted by the red line.</p

The relative mRNA expression of the stimulated cells and unstimulated controls at the 4h time point.

<p>Whiskers and outliers determined by the Tukey method. C = Control; Δ = PG343 biofilms; WT = W50 biofilms. Asterisk after cytokine name in the x-axis denotes statistical significance at p<0.05 between Δ and WT.</p

The composition of the W50 and PG343 biofilms.

<p>CFU ml^-1 normalized to the amounts of DNA extracted from each biofilm (n = 9 for both PG343 and W50 biofilms). p<0.01 for <i>F</i>. <i>nucleatum</i>, <i>V</i>. <i>dispar</i> and <i>P</i>. <i>gingivalis</i>. Boxes extend from 25th to 75th percentile, mid line being median. Whiskers and outliers plotted by the Tukey method. Asterisk before species name indicates statistical significance.</p