Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Stimulation and homogenization of the protoporphyrin IX endogenous production by photobiomodulation to increase the potency of photodynamic therapy.

Protoporphyrin IX (PpIX) is produced in the mitochondria and used as fluorescent contrast agent or photosensitizer after exogenous 5-aminolevulinic acid (ALA) delivery in cancer photodynamic detection and therapy (PDT). Although routinely used in the clinics, the stimulated production of PpIX is often insufficient and/or heterogeneous within the lesions, thereby limiting the PDT performances. Since photobiomodulation, which is based on the illumination of the tissues with sub-thermal radiometric conditions in the red or near-infrared, is known to stimulate the cell metabolism, we have optimized these conditions in vitro. Some of them lead to the homogenization and strong stimulation of the PpIX endogenous production. Interestingly, combined sequentially, PBM enhanced significantly the potency of PpIX-based PDT in vitro and in vivo in tumors grown on the chicken embryo chorioallantoic membrane. These results are in excellent agreement with other assays based on measurements of the cell survival/death, the production of reactive oxygen species, including singlet oxygen, and the mitochondrial membrane potential.

Comparative transcriptome analysis identifies genes associated with chlorophyll levels and reveals photosynthesis in green flesh of radish taproot.

The flesh of the taproot of Raphanus sativus L. is rich in chlorophyll (Chl) throughout the developmental process, which is why the flesh is green. However, little is known about which genes are associated with Chl accumulation in this non-foliar, internal green tissue and whether the green flesh can perform photosynthesis. To determine these aspects, we measured the Chl content, examined Chl fluorescence, and carried out comparative transcriptome analyses of taproot flesh between green-fleshed "Cuishuai" and white-fleshed "Zhedachang" across five developmental stages. Numerous genes involved in the Chl metabolic pathway were identified. It was found that Chl accumulation in radish green flesh may be due to the low expression of Chl degradation genes and high expression of Chl biosynthesis genes, especially those associated with Part Ⅳ (from Protoporphyrin Ⅸ to Chl a). Bioinformatics analysis revealed that differentially expressed genes between "Cuishuai" and "Zhedachang" were significantly enriched in photosynthesis-related pathways, such as photosynthesis, antenna proteins, porphyrin and Chl metabolism, carbon fixation, and photorespiration. Twenty-five genes involved in the Calvin cycle were highly expressed in "Cuishuai". These findings suggested that photosynthesis occurred in the radish green flesh, which was also supported by the results of Chl fluorescence. Our study provides transcriptome data on radish taproots and provides new information on the formation and function of radish green flesh.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

High ZnPP-forming food-grade lactic acid bacteria as a potential substitute for nitrite/nitrate to improve the color of meat products.

Zinc protoporphyrin IX (ZnPP)-forming food-grade lactic acid bacteria (LAB) were screened from various sources for their ability to improve the color of meat products. The effects of salt and nitrite on the ZnPP-forming ability of these bacteria were also investigated. Finally, these bacteria were applied in salt-added minced meat to assess their ability to improve the color. Twenty-five LAB were screened for their ZnPP-forming ability in pork. Most of the strains exhibited maximum growth anaerobically in 3% salt at 30 °C and grew well at pH 5.5 and 6.5. Moreover, 3% salt slightly retarded ZnPP formation; however, nitrite completely inhibited ZnPP formation in all the ZnPP-forming LAB. Thirteen LAB (avoiding duplication and non-food-grade) could form ZnPP in salt-added minced meat, resulting in improvement of the bright red color, high ZnPP autofluorescence, and increased fluorescence intensity. Finally, considering the safety, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, and Leuconostoc lactis were suggested as promising candidates to improve the color of meat products.

[Accidental feeding of the coccidiostat nicarbazin in layer breeder flocks - A case report]. / Versehentliche Fütterung von Legehennen-Elterntier-Herden mit dem Kokzidiostatikum Nicarbazin.

Following the accidental feeding of a compound feed containing the coccidiostat nicarbacin in layer breeder flocks (Lohmann Brown Classic), the birds displayed distinct clinical signs within a few hours. Mortality increased during the following 5 days, whereas laying performance and hatching rate of eggs during this period decreased markedly. Egg shell discoloration was observed as early as during the first day. As a consequence, an association between feeding of the coccidiostat nicarbacin and the observed symptoms was assumed. Recent studies indicate that Nicarbacin reduces the activity of aminolevulinic acid synthase type 1 (ALAS 1), which is responsible for the synthesis of protoporphyrin IX in the shell gland as main compound of brown egg shells. Reduced laying performance and increased mortality was likely due to nicarbacin-induced deregulated body temperature homeostasis and concomitant imbalances in acid-base status of the animals. The case reveals that the accidental feeding of nicarbacin to non-target animals such as laying hens and their parents may result in acute clinical symptoms. This highlights the necessity of appropriate care in handling feed additives and their premixes for specific non-target animals and should sensitize farmers and veterinarians.

Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Searching for high ZnPP-forming edible bacteria to improve the color of fermented meat products without nitrite/nitrate.

In order to improve the color of meat products by producing zinc protoporphyrin IX (ZnPP) in meat, we searched for edible bacteria with high ZnPP-forming ability. Eleven bacteria used in different animal products and 126 bacteria isolated from environmental and probiotic sources were assessed for their ability to form ZnPP. Many bacteria from both sources showed a high ZnPP-forming ability. Only three edible bacteria were identified from the 44 high ZnPP-forming isolates with 16S rRNA gene sequencing. High ZnPP-forming bacteria from both sources were inoculated in aseptic salt-added minced meat, and their ZnPP-forming abilities were evaluated. Lactococcus lactis, Leuconostoc mesenteroides, and Enterococcus faecium from environmental isolates produced a brighter red color, higher ZnPP autofluorescence and fluorescence intensity in salt-added minced meat than control. Furthermore, after heating, the color and ZnPP autofluorescence of the inoculated minced meat persisted to a degree. Therefore, it is possible to improve the color of meat products without nitrite/nitrate by using these promising ZnPP-forming edible bacteria.

Novel strategy to increase specificity of ALA-Induced PpIX accumulation through inhibition of transporters involved in ALA uptake.

BACKGROUND: Aminolevulinic acid-based photodynamic therapy (ALA-PDT) has emerged as a cancer treatment due to its high specificity and low side effects. In this study, we aimed to identify possible new drugs targeting transporters highly expressed in normal cells but not in cancer cells, to increase the specificity of ALA-PDT. METHOD: We used a total of seven cell lines, consisting of two gastric, three prostate, and two lung cell lines, for this purpose. siRNAs and inhibitors of these transporters were added, and PpIX production was evaluated using HPLC to examine the roles of transporters in ALA uptake. RESULTS: No correlation in the expression of transporters was observed among cell lines of the same origin. Two major findings were obtained: PEPT1 and PAT1 were expressed only in normal lung and prostate cells, respectively, but not in their cancerous counterparts. The inhibition of these transporters saw a significant decrease in PpIX production only in normal cells, but not in cancer cells. CONCLUSION: These findings show that the usage of drugs targeted specifically to highly expressed transporters in normal cells is essential for reducing PpIX accumulation in normal cells in order to increase the specificity of ALA-PDT in cancer.

Ferrochelatase activity of plant frataxin.

Frataxin plays a key role in cellular iron homeostasis of different organisms. It is engaged in several activities at the FeS cluster assembly machinery and it is also involved in heme biosynthesis. In plants, two genes encoding ferrochelatases (FC1 and FC2) catalyze the incorporation of iron into protoporphyrin IX in the last stage of heme synthesis in chloroplasts. Despite ferrochelatases are absent from other cell compartments, a remaining ferrochelatase activity has been observed in plant mitochondria. Here we analyze the possibility that frataxin acts as the iron donor to protoporphyrin IX for the synthesis of heme groups in plant mitochondria. Our findings show that frataxin catalyzes the formation of heme in vitro when it is incubated with iron and protoporphyrin IX. When frataxin is combined with AtNFS1 and AtISD11 the ferrochelatse activity is increased. These results suggest that frataxin could be the iron donor in the final step of heme synthesis in plant mitochondria, and constitutes an important advance in the elucidation of the mechanisms of heme synthesis in plants.

Sequentially self-assembled polysaccharide-based nanocomplexes for combined chemotherapy and photodynamic therapy of breast cancer.

Combination of chemotherapy and photodynamic therapy has emerged as a promising anticancer strategy. Polysaccharide-based nanoparticles are being intensively explored as drug carriers for different forms of combination therapy. In this study, novel multifunctional polysaccharide-based nanocomplexes were prepared from aldehyde-functionalized hyaluronic acid and hydroxyethyl chitosan via sequential self-assembly method. Stable nanocomplexes were obtained through both Schiff's base bond and electrostatic interactions. Chemotherapeutics doxorubicin and pro-photosensitizer 5-aminolevulinic acid were chemically conjugated onto the nanocomplexes via Schiff base linkage. Anti-HER2 antibody as targeting moiety was decorated onto the surface of nanocomplexes. The obtained near-spherical shaped nanocomplexes had an average size of 140 nm and a zeta potential of -24.6 mV, and displayed pH-responsive surface charge reversal and drug release. Active targeting strategy significantly enhanced the cellular uptake of nanocomplexes and combined anticancer efficiency of chemo-photodynamic dual therapy in breast cancer MCF-7 cells. These results suggested that the nanocomplexes had great potential for targeted combination therapy of breast cancer.

Design of Bifunctional Dendritic 5-Aminolevulinic Acid and Hydroxypyridinone Conjugates for Photodynamic Therapy.

Iron chelators have recently attracted interest in the field of photodynamic therapy (PDT) owing to their role in enhancement of intracellular protoporphyrin IX (PpIX) generation induced by 5-aminolevulinic acid (ALA) via the biosynthetic heme cycle. Although ALA is widely used in PDT, cellular uptake of ALA is limited by its hydrophilicity. In order to improve ALA delivery and enhance the PpIX production, several dendrimers incorporating both ALA and 3-hydroxy-4-pyridinone (HPO) were synthesized. The ability of the dendrimers to enter cells and be metabolized to the PpIX photosensitizer was studied in several human cancer cell lines. The dendrimers were found to be significantly more efficient than ALA alone in PpIX production. The higher intracellular PpIX levels showed a clear correlation with enhanced cellular phototoxicity following light exposure. Dendritic derivatives are therefore capable of efficiently delivering both ALA and HPO, which act synergistically to amplify in vitro PpIX levels and enhance PDT efficacy.

5-Aminolevulinic acid-mediated photodynamic therapy can target human glioma stem-like cells refractory to antineoplastic agents.

BACKGROUND: Glioblastoma (GBM) is a highly malignant lethal brain cancer. Accumulated evidence suggests that elevated resistance of GBM to both chemo- and radio-therapy is, at least in part, due to the presence of a small population of glioma stem cells (GSC). In the present study, we aimed to determine the sensitivity of GSCs to 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT). METHODS: For this purpose, we established GSC-enriched cell cultures (termed glioma stem-like cells or GSLCs) from A172 human GBM cell line. Under our cultivation conditions, GSLCs formed floating spheroid clusters that contained increased population of CD133/Sox2 expressing cells. Firstly, to compare the activity of protoporphyrin IX (PpIX) biosynthesis in the GSLCs and the parental A172 glioma cells, we examined the expression levels of biosynthesis enzymes and transporters for PpIX using qRT-PCR, and investigated the intracellular levels of PpIX with use of flow cytometry analysis. Then, we evaluated the sensitivity of these cells to ALA-PDT in vitro. Finally, to confirm the therapeutic impact of ALA-PDT on GSLCs with more clinically relevant model, we performed the same experiment using three different patient-derived glioma sphere lines, which cultivated them either in stem cell media or under differentiation conditions in the presence of serum. RESULTS AND CONCLUSION: GSLCs expressed higher mRNA levels of PpIX biosynthesis enzymes and its transporters PEPT1/2 and ABCB6, when compared to the parental A172 glioma cells. Consistently, flow cytometry analysis revealed that upon incubation with ALA, GSLCs accumulate a higher level of PpIX. Finally, we showed that GSLCs were more sensitive to ALA-PDT than the original A172 cells, and confirmed that all patient-derived glioma sphere lines also showed significantly increased sensitivity to ALA-PDT if cultivated under the pro-stem cell condition. Our data indicate that ALA-PDT has potential as a novel clinically useful treatment that might eliminate GBM stem cells that are highly resistant to current chemo- and radio-therapy.

Complete enzyme set for chlorophyll biosynthesis in Escherichia coli.

Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.

Assessment of safety of 5-aminolevulinic acid-mediated photodynamic therapy in rat brain.

BACKGROUND: Oral 5-aminolevulinic acid (ALA) induces biosynthesis/accumulation of the natural photo-sensitizer protoporphyrin IX (PpIX) in cancer cells. ALA is used widely in photodynamic diagnosis (PDD) and therapy (PDT) during malignant glioma surgery, but few studies have examined the effects of photodynamics plus ALA on normal brain tissue in vivo. We investigated the effects of ALA-mediated PDD and PDT on normal brain tissue. METHODS: We established a rat model in which the brain surface was irradiated through the skull by light-emitting diode (635 nm) after ALA administration. Using this model, we investigated the effects of various amounts of light irradiation with various ALA doses on brain tissue. RESULTS: Neurological symptoms developed with administration of ALA at 240 or 120 mg/kg accompanied by irradiation at 100 or 400 J/cm2, respectively. Dye leakage occurred due to disruption of the blood-brain barrier (BBB) at 90 mg/kg and 100 J/cm2, respectively. Thickness of the cortex increased significantly at 240 mg/kg and 400 J/cm2, respectively. The number of neurons appeared to decrease at 200 mg/kg plus 400 J/cm2, respectively, and there was an increase in the number of cells that were positive for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining. CONCLUSIONS: ALA-mediated PDT is safe at doses of 90 mg/kg or less followed by light irradiation of 100 J/cm2 in rat brains. At doses above this threshold, ALA-PDT led to irreversible BBB and brain damage in rats.

Scanning Fiber Endoscope Improves Detection of 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence at the Boundary of Infiltrative Glioma.

OBJECTIVE: Fluorescence-guided surgery with protoporphyrin IX (PpIX) as a photodiagnostic marker is gaining acceptance for resection of malignant gliomas. Current wide-field imaging technologies do not have sufficient sensitivity to detect low PpIX concentrations. We evaluated a scanning fiber endoscope (SFE) for detection of PpIX fluorescence in gliomas and compared it to an operating microscope (OPMI) equipped with a fluorescence module and to a benchtop confocal laser scanning microscope (CLSM). METHODS: 5-Aminolevulinic acid-induced PpIX fluorescence was assessed in GL261-Luc2 cells in vitro and in vivo after implantation in mouse brains, at an invading glioma growth stage, simulating residual tumor. Intraoperative fluorescence of high and low PpIX concentrations in normal brain and tumor regions with SFE, OPMI, CLSM, and histopathology were compared. RESULTS: SFE imaging of PpIX correlated to CLSM at the cellular level. PpIX accumulated in normal brain cells but significantly less than in glioma cells. SFE was more sensitive to accumulated PpIX in fluorescent brain areas than OPMI (P < 0.01) and dramatically increased imaging time (>6×) before tumor-to-background contrast was diminished because of photobleaching. CONCLUSIONS: SFE provides new endoscopic capabilities to view PpIX-fluorescing tumor regions at cellular resolution. SFE may allow accurate imaging of 5-aminolevulinic acid labeling of gliomas and other tumor types when current detection techniques have failed to provide reliable visualization. SFE was significantly more sensitive than OPMI to low PpIX concentrations, which is relevant to identifying the leading edge or metastasizing cells of malignant glioma or to treating low-grade gliomas. This new application has the potential to benefit surgical outcomes.

The influence of different illumination parameters on protoporphyrin IX induced cell death in squamous cell carcinoma cells.

BACKGROUND: Topical photodynamic therapy (PDT) is a highly effective therapy especially for extended cancerized fields of the skin. Whenever extended fields are treated pain management is advisable. Light source mediated pain management can be performed by reducing fluence rates, as long as this does not compromise efficacy. METHODS: Two squamous cell carcinoma cell lines (A431 and SCC-13) were subjected to in vitro PDT using two different ALA concentrations and synthesis intervals and protoporphyrin IX (PpIX) synthesis was assessed. Two total light doses (6 J/cm2 and 37 J/cm2) were applied at three different fluence rates and cell viability was measured using the MTS-test. RESULTS: Both cell lines synthetized PpIX at different kinetics. A431 cells produced a maximum 28.6 nmol/l PpIX, while SCC-13 reached only a production of 8.7 nmol/l. Illumination reduced cell viability depending on PpIX content and light dose. When a lower light dose (6 J/cm2) was applied, only the combination with the highest PpIX content was effective in A431 cells and no effect could be detected in SCC-13 cells. With a light dose of 37 J/cm2, lower PpIX amounts became effective in A431 and cell death could be induced in SCC-13 cells. Light fluence rate had no differential effect in this setup. CONCLUSIONS: In both, A431 and SCC-13 cells, total light dose is a key factor for photodynamic efficacy. Additionally, our results hint towards a threshold concentration of PpIX upon which a drastic loss of viability occurs. Light fluence rate in the analyzed range is not a limiting factor of photodynamic cytotoxicity. This may allow for the clinical implementation of low fluence rate protocols for pain management without compromising efficacy.

Liaison between heme metabolism and bioenergetics pathways-a multimodal elucidation for early diagnosis of oral cancer.

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in 'redox ratio' were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway (viz. 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes (viz. FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells (viz. fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method.

Photodynamic therapy using 5-aminolevulinic acid triggered DNA damage of adenocarcinoma breast cancer and hepatocellular carcinoma cell lines.

Targeting cancer cells with photosensitizer (PS) excited by appropriate laser irradiation to release singlet oxygen as a photodynamic therapy (PDT) remains a challenge. This research aimed to assess the cytogenetic potential of 5-aminolevulinic acid (5-ALA) activated with laser irradiation (5-ALA/PDT) to damage the intact DNA of adenocarcinoma breast cancer cell line (MCF-7) and hepatocellular carcinoma cell line (HepG2). Cancer cells were treated with 0.5 and 1 mM 5-ALA for 4 h, the precursor of the photochemical protoporphyrin IX (PpIX), and then exposed to laser irradiation at 633 nm and 0.25 W for 4 min before incubation for 24 h. Cytotoxicity of cancer cells was assessed using trypan blue exclusion assay. Genotoxicity was recorded by micronucleus test and comet assay. Both 5-ALA and laser irradiation, separately, were nontoxic on cancer cell lines, however, 5-ALA/PDT enhanced cell death in a concentration-dependent manner. Also, 5-ALA/PDT generated high percentages of micronuclei in MCF-7 and HepG2 cell lines as recorded in binucleated cells. Similarly, the mean percentages of DNA damage and tail moment ratio were intensified extremely in cancer cell lines treated with 5-ALA/PDT rather than non-treated cells or cells treated by 5-ALA or laser irradiation separately. In conclusion, the singlet oxygen of 5-ALA targets DNA of cancer cells when activated by laser irradiation. Therefore, photodynamic therapy is an applicable process to damage DNA effectively during M-phase and prohibit cancer cells proliferation.

Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages.

Nitrite is commonly used in meat products due to its plural technological advantages. However, it is controversial because of its detrimental side effects on health. Within the context of nitrite reduction, zinc protoporphyrin IX (Zn(II)PPIX) formation in meat products as natural red colouring agent has been suggested. This investigation presents the evaluation of naturally occurring pigments, namely Zn(II)PPIX, protoporphyrin IX (PPIX) and heme in nitrite-free dry fermented sausages in function of time, meat source (pork, horsemeat and a combination of both meat sources) and pH condition. In function of time, Zn(II)PPIX and PPIX were formed and heme content decreased. Higher pH conditions promoted Zn(II)PPIX and PPIX formation, whereas the influence of pH on heme was less clear. The use of horsemeat also promoted Zn(II)PPIX formation. Moreover, even similar amounts were formed when it was combined with pork. Product redness, however, could not be related to Zn(II)PPIX formation.