Tomato Phytonutrients Balance UV Response: Results from a Double-Blind, Randomized, Placebo-Controlled Study.

BACKGROUND: Our previous double-blinded, placebo-controlled cross-over study indicated that a nutritional supplement named lycopene-rich tomato nutrient complex (TNC) can protect from UVA1-induced (340-400 nm) and UVA- (320-400 nm)/UVB-induced (280-320 nm) upregulation of molecular markers associated with oxidative stress, inflammation, and ageing. OBJECTIVES: in the current double-blind, randomized, placebo-controlled multicenter study, we analyze whether a similar, synergistic carotenoid-rich TNC can protect from broadband UVB-induced threshold erythema formation assessed as increase in minimal erythemal dose (MED) reading, the intensity of erythema formation, and the upregulation of molecular markers associated with inflammation and immunosuppression, and whether this correlates with carotenoid blood levels. METHODS: One hundred and forty-nine healthy volunteers were randomized to two groups and subjected to a 5-week washout phase, followed by a 12-week treatment phase receiving either 15 mg lycopene, 5.8 mg phytoene and phytofluene, 0.8 mg ß-carotene, 5.6 mg tocopherols from tomato extract, and 4 mg carnosic acid from rosemary extract per day or placebo made from medium-chain triglycerides. At the end of each phase, MED determination, UVB irradiation, chromametry, biopsies, and blood samples were undertaken. RESULTS: The active supplement was well tolerated. Interestingly, no significant difference was seen in the MED between the active-supplement and placebo groups, as determined by visual grading by expert assessors. Of note, the carotenoid-containing supplement significantly protected against UVB-induced erythema formation measured as Δa* after the intervention minus Δa* after the washout phase as compared to the placebo. Moreover, intake of the active supplement significantly protected against UVB-induced upregulation of IL6 and TNFα as compared with the intake of placebo. Lastly, carotenoid plasma levels were significantly increased. CONCLUSION: This well-tolerated carotenoid-containing supplement significantly protected against UVB-induced erythema formation and upregulation of proinflammatory cytokines in healthy volunteers.

Quantifying Direct DNA Damage in the Basal Layer of Skin Exposed to UV Radiation from Sunbeds.

Nonmelanoma and melanoma skin cancers are attributable to DNA damage caused by ultraviolet (UV) radiation exposure. One DNA photoproduct, the cyclobutane pyrimidine dimer (CPD), is believed to lead to DNA mutations caused by UV radiation. Using radiative transfer simulations, we compare the number of CPDs directly induced by UV irradiation from artificial and natural UV sources (a standard sunbed and the midday summer Mediterranean sun) for skin types I and II on the Fitzpatrick scale. We use Monte Carlo radiative transfer (MCRT) modeling to track the progression of UV photons through a multilayered three dimensional (3D) grid that simulates the upper layers of the skin. By recording the energy deposited in the DNA-containing cells of the basal layer, the number of CPDs formed can be quantified. The aim of this work was to compare the number of CPDs formed in the basal layer of the skin and by implication the risk of developing cancer, as a consequence of irradiation by artificial and natural sources. Our simulations show that the number of CPDs formed per second during sunbed irradiation is almost three times that formed during solar irradiation.

Carbonate Anion Radical Generated by the Peroxidase Activity of Copper-Zinc Superoxide Dismutase: Scavenging of Radical and Protection of Enzyme by Hypotaurine and Cysteine Sulfinic Acid.

Copper-zinc superoxide dismutase (SOD) is considered one of the most important mammalian antioxidant defenses and plays a relevant role due to its main function in catalyzing the dismutation of superoxide anion to oxygen and hydrogen peroxide. However, interaction between SOD and H2O2 produced a strong copper-bound oxidant (Cu(II)•OH) that seems able to contrast the self-inactivation of the enzyme or oxidize other molecules through its peroxidase activity. The bicarbonate presence enhances the peroxidase activity and produces the carbonate anion radical (CO3•-). CO3•- is a freely diffusible reactive species capable of oxidizing several molecules that are unwieldy to access into the reactive site of the enzyme. Cu(II)•OH oxidizes bicarbonate to the CO3•-, which spreads out of the binding site and oxidizes hypotaurine and cysteine sulfinic acid to the respective sulfonates through an efficient reaction. These findings suggest a defense role for sulfinates against the damage caused by CO3•- . The effect of hypotaurine and cysteine sulfinic acid on the CO3•--mediated oxidation of the peroxidase probe ABTS to ABTS cation radical (ABTS•+) has been studied. Both sulfinates are able to inhibit the oxidation of ABTS mediated by CO3•-. The effect of hypotaurine and cysteine sulfinic acid against SOD inactivation by H2O2 (~42% protection of enzyme activity) has also been investigated. Interestingly, hypotaurine and cysteine sulfinic acid partially avoid the H2O2-mediated SOD inactivation, suggesting that the two sulfinates may have access to the SOD reactive site and preserve it by reacting with the copper-bound oxidant. In this way hypotaurine and cysteine sulfinic acid not only intercept CO3•- which could move out from the reactive site and cause oxidative damage, but also prevents the inactivation of SOD.

Daylight photodynamic therapy in Scotland.

Chronic sun-induced dysplastic skin changes (actinic keratoses) are extremely common in fair-skinned people in Scotland. These changes are a major cause of morbidity and may develop into skin cancer. Actinic keratoses are often extensive and pose a therapeutic challenge as field-directed treatment is required for chronic disease management. One such treatment approach is hospital-based photodynamic therapy, which is a well-established treatment in Scotland for actinic keratoses, using a photosensitiser pro-drug and red LED light irradiation. However, photodynamic therapy using daylight as the activating light source is increasingly and effectively used in continental Europe, but had not been explored in Scotland until we initiated this in 2013. We report our experience of daylight photodynamic therapy in 64 patient-treatment courses and demonstrate that this can be an effective, well-tolerated treatment, which is liked by patients. Our most recent data show that most patients (73%) achieved clearance or at least a good response to treatment and had high levels of satisfaction with daylight photodynamic therapy. Daylight exposure measurements indicated that treatment is feasible in Scotland between April to September. Daylight photodynamic therapy is an important advancement in treatment options for Scottish patients with extensive pre-cancerous field changes and provides opportunities for home-based treatment and increased efficiency of photodynamic therapy services.

A quantitative study of in vivo protoporphyrin IX fluorescence build up during occlusive treatment phases.

BACKGROUND: Topical photodynamic therapy (PDT) is a non-invasive light based therapy used to treat non-melanoma skin cancer (NMSC) and dysplasia. During PDT, the light sensitive molecule protoporphyrin IX (PpIX) is activated, resulting in the production of singlet oxygen, which subsequently leads to cell death. PpIX is metabolised from a topically applied pro-drug and the strong fluorescence signal associated with PpIX can be utilised as an indicator of the amount of PpIX present within the tumour tissue. In this work we measure the build up PpIX during the occlusive treatment phase and investigate how the PpIX production rate is affected by different lesion and patient characteristics. METHODS: Fluorescence measurements were used to investigate the build up of PpIX within the tumour tissue during the 3h long occlusive treatment prior to irradiation. The study included in vivo measurements of 38 lesions from 38 individual patients. Actinic keratosis (AK) and basal cell carcinoma (BCC) were the lesion types included in this study. The resulting data from the study was analysed using generalised linear mixed effects models. RESULTS: It was found that the surface fluorescence signal linearly increased with occlusive treatment time. The predictive models suggest that there is a significant difference in PpIX production between lesion location, however no significant difference is demonstrated between different lesion types, gender and skin type. CONCLUSIONS: The study extends and supports previous knowledge of PpIX production during the occlusive treatment phase.

Photodynamic diagnostic ureterorenoscopy: A valuable tool in the detection of upper urinary tract tumour.

BACKGROUND: Photodynamic diagnosis increases the detection rate and hence decreases recurrence rates of urothelial cancer (UC) of the bladder. This technique has been implemented in the upper urinary tract and like in the bladder, has shown to increase the detection rate of urothelial lesions. OBJECTIVES: To determine the sensitivity, specificity, and detection rates for photodynamic diagnostic flexible ureterorenoscopy (PDD-FURS) and white light ureterorenoscopy (WL-FURS). Design between 2009 and 2013, PDD-FURS was performed within 106 Upper urinary tract (UUT) Units (Mean age-72.6±9.5). Indications for the procedure included abnormal upper urinary tract on imaging, normal flexible cystoscopy with abnormal urine cytology, endoscopic treatment and follow-up of UUT UC. Oral 5-aminolevulinic acid was used as the photosensitizer administered 3-4 h pre-operatively. RESULTS: 48 lesions were detected, of which 95.8% (46/48) where visualised by PDD-FURS compared to 47.9% (23/48) shown by WL-FURS (P<0.0001). PDD-FURS detected significantly more carcinoma in situ (CIS) or dysplasia lesions than WL-FURS (93.75% (15/16) vs. 18.75% (3/16), respectively, (P=0.0006)). Furthermore, PDD-FURS detected significantly more UC lesions than WL-FURS (96.9% (31/32) vs. 62.5% (20/32) (P=0.007)). PDD-FURS was more sensitive (95.8; range: 85.7-99.5) than WL-FURS (53.5; range: 37.7-68.8) in detecting UUT-UC (P<0.0001). There was no difference (P=0.716) in the specificity between PDD-FURS (96.6; range: 88.1-99.6) and WL-FURS (95.2; range: 86.7-99). CONCLUSIONS: Our results PDD-FURS with oral 5-ALA as photosensitizer suggest higher sensitivity and detection rate of urothelial tumours than WL-FURS, with a good safety profile. In our series, PDD-FURS enhanced the visualisation of flat lesions, such as CIS and dysplasia that otherwise would have been missed.

Development of a handheld fluorescence imaging device to investigate the characteristics of protoporphyrin IX fluorescence in healthy and diseased skin.

BACKGROUND: Topical Photodynamic therapy (PDT) is an effective treatment for superficial non-melanoma skin cancers (NMSC) and dysplasia. During PDT light activates the photosensitiser (PpIX), metabolised from a topical pro-drug. A combination of PpIX, light and molecular oxygen results in inflammation and cell death. However, the outcomes of the treatment could be better. Insufficient biosynthesis of PpIX may be one of the causes of incomplete response or recurrence. Measuring surface fluorescence is usually employed as a means of studying PpIX formation. The aim of this work was to develop a device and a method for convenient fluorescence imaging in clinical settings to gather information on PpIX metabolism in healthy skin and NMSC with a view to improving PDT regimes. METHODS: A handheld fluorescence camera and a time course imaging method was developed and used in healthy volunteers and patients diagnosed with basal cell carcinoma (BCC) and actinic keratosis (AK). The photosensitiser (precursor) creams used were 5-aminolaevulinic acid (ALA; Ameluz(®)) and methyl aminolevulinate (MAL; Metvix(®)). Pain was assessed using a visual analogue score immediately after the PDT. RESULTS: Fluorescence due to PpIX increases over three hours incubation in healthy skin and in lesional BCC and AK. Distribution of PpIX fluorescence varies between the lesion types and between subjects. There was no significant correlation between PpIX fluorescence characteristics and pro-drug, diagnosis or pain experienced. However, there was a clear dependence on body site. CONCLUSION: The device and the method developed can be used to assess the characteristics of PpIX fluorescence, quantitative analysis and time course. Our findings show that body site influences PpIX fluorescence which we suggest may be due to the difference in skin temperature at different body sites.

Laser-induced autofluorescence spectroscopy: can it be of importance in detection of bladder lesions?

BACKGROUND: Non-muscle invasive bladder cancer can be missed during white light endoscopy in up to 50% of cases. We aimed to test whether or not we could find a difference between benign and cancerous tissue wavelengths using laser induced autofluorescence spectroscopy can increase cancer detection. MATERIALS AND METHODS: We analysed 67 tissue samples using spectral analysis. The WavSTAT (Spectra Science) optical biopsy device was used to record fluorescence spectra from biopsied tissue enabling calculation of an AUC for each spectrum, a measure of the mean spectral wavelength (λ¯ (nm)) and a dimensionless fluorescence ratio. Mann-Whitney test was used to compare the two groups. RESULTS: We found that 49.3% (33/67) of the tissue was benign, 44.8% (30/67) was CIS/cancerous tissue, and the remaining 4/67 samples were atypia (2) and dysplasia (2). The median AUC for the benign tissue was 19.53 (interquartile range [IQR]: 5.35-30.39) and that for CIS/cancerous tissue was 7.05 (IQR: 2.89-14.24) (P=0.002). The median wavelengths for the benign tissue and malignant tissue were 502.4nm (IQR: 500.3-504.3nm) and 505.2nm (IQR: 502.1-513.2nm), respectively (P=0.003). The median fluorescence ratio was 0.080 (IQR: 0.070-0.088) for benign tissue and 0.096 (IQR: 0.079-0.221) for CIS/cancerous tissue (P=0.002). CONCLUSIONS: We found statistical differences between the median AUC calculations and median wavelengths for the benign and cancerous tissue. We also found a statistical difference between the fluorescence ratios between the two tissue types. There seems to be a role for optical spectroscopy in verifying bladder lesions.

The effect of 222-nm UVC phototesting on healthy volunteer skin: a pilot study.

BACKGROUND: Frequent topical antiseptic use to hands is now common in healthcare and other work environments. Inevitably, the use of such antiseptics will present an occupational risk for irritancy and allergic dermatitis. New, less irritant and even non-chemical antimicrobial approaches are under investigation. METHODS: A Sterilray disinfectant source (222 nm) conventionally used to sterilize equipment and work surfaces was assessed for tolerability in human skin. Using an escalating dosage study methodology, four skin phototype I and II healthy volunteers had their minimal erythema dose (MED) determined. Punch biopsies of irradiated sites were stained for cyclobutane pyrimidine dimers (CPD). The degree of CPD was compared with that in biopsies from unexposed skin and from areas exposed to UVB (280-315 nm) radiation. RESULTS: Calibrated spectral measurements revealed emission at a peak wavelength of 222 nm with 97% emission at wavelengths less than 250 nm. At low doses below the threshold bacteriostatic effect, the source was capable of inducing both erythema and CPD formation in human skin. In two individuals, cells in the basal layer were not shielded by the overlying tissue as indicated by the presence of CPD. CONCLUSION: The source showed an erythemogenic or CPD potential at lower doses than those required to reach the reported threshold bacteriostatic effect.

Photodynamic diagnostic ureterorenoscopy with orally administered 5-aminolaevulinic acid as photosensitiser: how I do it.

OBJECTIVE: To explain our use of photodynamic diagnostic ureterorenoscopy, we provide a detailed description of the choice of photosensitiser, equipment needed, a safety profile, and pointers on our technique. TECHNIQUE: Patients are given oral 5-aminolaevulinic acid (5-ALA) as a photosensitiser 3-4 h pre-operatively, and by using a Xenon blue light source, an eyepiece which blocks light below 450 nm which is fitted onto the ureterorenoscope, we are able to conduct a thorough photodiagnosis of the upper urinary tract. CONCLUSION: Our technique of photodynamic diagnostic ureterorenoscopy has shown that the sensitivity, specificity and detection rates of upper urinary tract lesions can be significantly increased with the use of oral 5-ALA. Therefore, we provide a detailed explanation of the use of oral 5-ALA photosensitiser, indications and contraindications of the technique in addition to equipment used and potential complications of the procedures.

UV emissions from low energy artificial light sources.

Energy efficient light sources have been introduced across Europe and many other countries world wide. The most common of these is the Compact Fluorescent Lamp (CFL), which has been shown to emit ultraviolet (UV) radiation. Light Emitting Diodes (LEDs) are an alternative technology that has minimal UV emissions. This brief review summarises the different energy efficient light sources available on the market and compares the UV levels and the subsequent effects on the skin of normal individuals and those who suffer from photodermatoses.

Comparison of intraoperative fluorescence and MRI image guided neuronavigation in malignant brain tumours, a prospective controlled study.

INTRODUCTION: MBT carry poor prognosis and more than 80% of MBT recur locally within 2 cm of the resection margin because of inadequate surgical removal. A number of techniques have been implemented in recent years to improve surgical removal of MBT with variable success. We examined two methods commonly used to resect MBT to establish which one offered the best chances of gross total removal; MRI guided technology and ALA-induced fluorescence. PATIENTS AND METHODS: Twenty consecutive patients diagnosed with MBT were included in this study. They were given 20mg ALA per kg body weight 3h before anaesthesia orally mixed in water. Surgery was planned using preoperative enhanced MPR age images. Surgery was executed using the Stealth Station image guidance system and ALA-induced fluorescence microsurgical techniques. During surgery the intensity of fluorescence was graded into red, pink or blue. The intensity of fluorescence was also measured using pulsed 405 nm laser and a compact spectrometer using a touch probe directly placed on the tissue. The extent of tumour invasion was assessed intraoperatively using standard white light, blue light and spectroscopic measurements. Postoperative enhanced MRI was used to assess the extent of resection and the volume of residual tumour was measured. RESULTS: There were six newly diagnosed GBM, eight recurrent GBM, one oligodendroglioma (ODG) and five metastases (MET). On enhanced MRI, the mean diameter of new GBM, recurrent GBM, ODG and MET was 2.3 cm, 2.3 cm, 1.5 cm, and 2.3 cm respectively. Under the blue light, the mean diameter of new GBM, recurrent GBM, ODG and MET was 2.9 cm, 3 cm, 1.5 cm and 2.3 cm respectively. The results of quantitative measurements of fluorescence ratios revealed that red fluorescence corresponded to 5.9-11.6 (solid tumour on histology), and pink fluorescence measured 0.8-1.9 (infiltrating edge of tumour on histology). When we compared the maximum tumour diameter of GBM we found on average it was 10mm wider on spectroscopy compared to standard white light microscopy and 6mm wider than what the enhanced MRI demonstrated. CONCLUSIONS: Fluorescence technology revealed that GBMs are wider than the enhanced MRI had demonstrated, while MET enhanced MRI was similar in size to fluorescence. Furthermore, solid tumour can be identified intraoperatively and can be measured using fluorescence and spectroscopy techniques and it can be removed safely. Infiltrating tumour can also be identified intraoperatively using this technology and can be removed in non-eloquent areas to maximise surgical resection.

Differential sensitivity in cell lines to photodynamic therapy in combination with ABCG2 inhibition.

BACKGROUND: ABCG2 is an ATP-binding cassette transporter protein which has a role in the regulation of endogenous protoporphyrin IX (PpIX) levels. OBJECTIVE: To understand the influence of ABCG2 on porphyrin-based photodynamic therapy (PDT) and fluorescence diagnosis (FD), we examined the role of endogenous ABCG2 in four human cell lines from the epidermis (HaCaT keratinocytes), oesophagus (OE19 adenocarcinoma), brain (SH-SY5Y neuroblastoma) and bladder (HT1197 carcinoma). METHODS: Cells were incubated with ALA or MAL in the presence or absence of the ABCG2 activity inhibitor Ko-143. Porphyrin accumulation was detected by spectrofluorimetric analysis and high performance liquid chromatography (HPLC) with porphyrin localisation observed by confocal laser scanning microscopy. PDT efficacy was assessed 24h post irradiation (1.5J/cm(2) red light) by the neutral red (NR) assay. RESULTS: We show cell-specific differences when Ko-143 was co-incubated with ALA or, in particular with, MAL. Enhanced PDT-induced cell kill was shown in HaCaT, OE19 and HT1197 cells, but not SH-SY5Y cells and could be explained by porphyrin accumulation and expression of ABCG2. We have also found that despite high levels of intracellular PpIX, the OE19 cells were protected from phototoxic cell death by PpIX compartmentalisation. This could be reversed by Ko-143. CONCLUSION: The results from this study show a possible cause of reduced sensitivity to ALA/MAL-PDT, with a potential solution to overcome this effect in certain tissue types. The potential to improve PDT with Ko-143 remains promising.

Diagnosis of upper urinary tract tumours: is photodynamic diagnosis assisted ureterorenoscopy required as an addition to modern imaging and ureterorenoscopy?

OBJECTIVES: We aimed to assess the diagnostic accuracy of photodynamic diagnostic ureterorenoscopy (PDD-FURS) in detection of UUT-TCC in comparison with CT Urogram (CTU) and WL-FURS. MATERIAL AND METHODS: Between June 2009 and August 2011, 30 patients underwent PDD-FURS after CTU for suspicion of UUT-TCC. Ureterorenoscopy was performed for abnormal upper urinary tract on imaging. Oral 5-Aminolevulinic Acid (5-ALA) was used as a photosensitizer. All procedures were performed by single endourologist experienced in photodynamic diagnosis. The sensitivity, specificity, and detection rate of WL-FURS, PDD-FURS and CTU were calculated using the Meta-DiSc v1.4 programme. P values <0.05 were considered significant. RESULTS: PDD-FURS detected more UUT-TCCs than CTU or WL-FURS (94% (16/17) vs. 76.5% (13/17) vs. 82% (14/17) respectively). PDD-FURS was not significantly more sensitive than CTU and WL-FURS to detect UUT-TCC (0.94 (95% CI: 0.71-0.99) vs. 0.82 (95% CI: 0.57-0.96) vs. 0.81 (95% CI: 0.54-0.96) respectively; PDD-FURS vs. CTU: P=0.249; PDD-FURS vs. WL-FURS: P=0.277). There was no difference in the specificity between PDD-FURS and WL-FURS (1.0 (95% CI: 0.75-1.0) and 1.0 (95% CI: 0.75-1.0) respectively) (P=1), while PDD-FURS was significantly more specific than CTU (CTU: 0.21 (95% CI: 0.05-0.51) (P<0.001). PDD-FURS picked up 3 cases of CIS, which was not seen on WL-FURL and CTU. CONCLUSION: Oral 5-ALA induced PDD-FURS has a high sensitivity and specificity to detect lesions and a higher detection rate to diagnose UUT-TCC than WL-FURS and CTU. It appears to be the only tool to visualise UUT CIS lesions.

Modelling fluorescence in clinical photodynamic therapy.

Understanding the interactions of non-ionizing radiation with living organisms has been the focus of much research over recent decades. The complex nature of these interactions warrants development of theoretical and experimental studies to gain an insight into predicting and monitoring the success of photodynamic therapy (PDT) protocols. There is a major impetus towards evidence-based recommendations for patient diagnosis, treatment and management. Knowledge of the biophysical aspects of PDT is important for improving dosimetry protocols. Fluorescence in clinical PDT may be used to detect and diagnose pre-malignant and malignant conditions, while photobleaching can monitor changes in fluorescence during treatment. Combining empirical fluorescence photobleaching clinical data with computational modelling enables clinical PDT dosimetry protocols to be investigated with a view to optimising treatment regimes. We will discuss how Monte Carlo radiation transfer (MCRT) modelling has been intercalated in the field of fluorescence detection and PDT. In this paper we highlight important aspects of basic research in PDT by reporting on the current utilisation of fluorescence in clinical PDT from both a clinical and theoretical perspective. Understanding and knowledge of light propagation in biological tissue from these perspectives should have a positive impact on treatment planning.