Studies of the metabolism and the toxicity of N-Methylformamide and related amides

The hepatotoxicity of the industrial solvent and investigational anti-tumour agent N-methylformamide (NMF, HOCNHCH3) and several structural analogues was assessed in mice. NMF and its ethyl analogue (NEF) were equipotent hepatotoxins causing extensive centrilobular necrosis and damage to the gall bladder. Pretreatment of mice with SKF525A did not influence the toxicity of these N-alkylformamides. Replacement of the formyl hydrogen of NMF with deuterium or methyl significantly reduced its hepatotoxicity. An in vitro model for the study of the toxicity and metabolism of N-alkylformamides was developed using isolated mouse hepatocytes. The cytotoxicity of NMF in vitro was concentration-dependent with maximal toxicity being achieved at concentrations of 5mM or above. The cytotoxic potential of related amides correlated well with their in vivo hepatotoxic potential. Pretreatment of mice with buthionine sulphoximine (BSO), which depleted hepatocytic levels of glutathione to 15% of control values, exacerbated the cytotoxicity of NMF towards the hepatocytes. NMF (1mM or above), incubated with isolated mouse hepatocytes, depleted intracellular glutathione levels to 26% of control values within 4h. Depletion of glutathione was quantitatively matched by the formation of a carbamoylating metabolite. Metabolism was dependent on the concentration of NMF and was drastically reduced in incubations of hepatocytes isolated from mice pretreated with BSO. The carbamoylating metabolite, S-(N-methylcarbamoyl)-glutathione (SMG), was identified in vitro using FAB-MS. The generation of SMG was subject to a large primary H/D kinetic isotope effect when the formyl hydrogen was replaced with deuterium. Likewise, glutathione depletion and metabolite formation were reduced or abolished by the deuteration or methylation of the formyl moiety of NMF. NEF, like NMF, depleted hepatocytic glutathione levels and was metabolised to a carbamoylating metabolite. Radioactivity derived from 14C-NMF and 14C-NEF, labelled in the alkyl moieties, was found to be irreversibly associated with microsomal protein on incubation in vitro. Binding was dependent on the presence of NADPH and was mostly abolished in the presence of reduced glutathione. SKF525A failed to influence the binding.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

Discrimination of healthy and cancer cells of the bladder by metabolic state, based on autofluorescence

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD ("redox ratio") can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

Computational model of bladder tissue based on its measured optical properties

Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a "generic" computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system "LAKK-M" was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

Changes in autofluorescence based organoid model of muscle invasive urinary bladder cancer

Muscle invasive urinary bladder cancer is one of the most lethal cancers and its detection at the time of transurethral resection remains limited and diagnostic methods are urgently needed. We have developed a muscle invasive transitional cell carcinoma (TCC) model of the bladder using porcine bladder scaffold and the human bladder cancer cell line 5637. The progression of implanted cancer cells to muscle invasion can be monitored by measuring changes in the spectrum of endogenous fluorophores such as reduced nicotinamide dinucleotide (NADH) and flavins. We believe this could act as a useful tool for the study of fluorescence dynamics of developing muscle invasive bladder cancer in patients.

Optical redox ratio and endogenous porphyrins in the detection of urinary bladder cancer:a patient biopsy analysis

Bladder cancer is among the most common cancers in the UK and conventional detection techniques suffer from low sensitivity, low specificity, or both. Recent attempts to address the disparity have led to progress in the field of autofluorescence as a means to diagnose the disease with high efficiency, however there is still a lot not known about autofluorescence profiles in the disease. The multi-functional diagnostic system "LAKK-M" was used to assess autofluorescence profiles of healthy and cancerous bladder tissue to identify novel biomarkers of the disease. Statistically significant differences were observed in the optical redox ratio (a measure of tissue metabolic activity), the amplitude of endogenous porphyrins and the NADH/porphyrin ratio between tissue types. These findings could advance understanding of bladder cancer and aid in the development of new techniques for detection and surveillance.