<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. The specific aim of this project was to study if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells and to do this with dose and time responses in focus. In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 in five different concentrations, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that regardless of dose or time, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP.</p>

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. This is a follow-up to an earlier study, and the specific aim of this project was to find out if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells during long-term treatment (up to 48 hours). In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 of the concentration 10-8 M, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that despite of the rather high dose used, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP, regardless of time.</p>

Σκοπός της παρούσας διατριβής ήταν η μελέτη της σκληρωτιακής διαφοροποίησης στις τέσσερις βασικές μορφές της στους μυκηλιακούς μύκητες S. rolfsii, S. sclerotiorum, R. solani και S. minor σε σχέση με το οξειδωτικό στρες. Ως μάρτυρες για τους υπό μελέτη μύκητες χρησιμοποιήθηκαν αντίστοιχα μη σκληρωτιογόνα στελέχη. Τα πιο σημαντικά υπεροξείδια που συμβάλλουν στην δημιουργία οξειδωτικού στρες είναι: α) η ρίζα του σουπεροξειδίου (O2•-), που είναι το πρωταρχικό αίτιο του οξειδωτικού στρες, β) το υπεροξείδιο του υδρογόνου (H2O2), που κυρίως προκύπτει από την ένωση δύο μορίων O2•- (αντίδραση αυτοοξειδοαναγωγής) και γ) τα λιπιδικά υδροϋπεροξείδια (LOOH), που είναι από τα πρώιμα αποτελέσματα του οξειδωτικού στρες, λόγω της προσβολής των πολυακόρεστων λιπαρών οξέων. Επειδή λοιπόν στην διεθνή βιβλιογραφία δεν υπήρχε κατάλληλη μεθοδολογία για τον προσδιορισμό του O2•-, αναπτύχθηκε μια νέα μέθοδος εφαρμόσιμη εκτός από τους υπό μελέτη μύκητες, σε όλους τους οργανισμούς. Επιπλέον, χρησιμοποιήθηκε η ποσοτικοποίηση της συγκέντρωσης των βασικών ενζύμων που σχετίζονται με τα προαναφερθέντα υπεροξείδια τα οποία είναι: α) η δισμουτάση του O2•- (SOD), που επιταχύνει την αντίδραση αυτοοξειδοαναγωγής, β) η καταλάση (CAT), που καταναλώνει το H2O2, γ) οι μη εξειδικευμένες υπεροξειδάσες (NSPX) που χρησιμοποιούν το H2O2 και τα LOOH σαν υπόστρωμα για να οξειδώνουν άλλα μόρια και δ) η οξειδάση της ξανθίνης (ΧΟ), που ανιχνεύεται για πρώτη φορά σε μύκητες και είναι υπεύθυνη μεταξύ άλλων και για την παραγωγή του O2•- και H2O2 στους οργανισμούς. Ακόμη, έγινε εκτίμηση της οξειδωτικής καταστροφής των μεμβρανικών λιπιδίων με τη μέτρηση των αλδεϋδικών παραγώγων της υπεροξείδωσης των λιπιδίων (MDA). Τέλος, χρησιμοποιήθηκαν οι εξής εξωγενείς τροποποιητές των παραπάνω μορίων: α) μιμητές της SOD β) H2O2 γ) υδροϋπεροξείδιο του κουμενίου (λιπιδικό υδροϋπεροξείδιο) και δ) αμινοτριαζόλη (αναστολέας της CAT). Τα αποτελέσματα της μελέτης επιβεβαίωσαν την αναγκαιότητα της ανάπτυξης της νέας μεθόδου ποσοτικοποίησης του O2•-. Συγκεκριμένα, δείχθηκε ότι οι τέσσερις μορφές σκληρωτιακής διαφοροποίησης εξαρτώνται άμεσα από το οξειδωτικό στρες, και ότι οι σχετιζόμενοι με αυτό παράγοντες που εξετάστηκαν σε αυτή τη μελέτη μεταβάλλονται με διαφορετικό τρόπο σε κάθε μορφή σκληρωτιακής διαφοροποίησης. Τα βασικότερα συμπεράσματα αυτής της μελέτης είναι: α) το O2•- φαίνεται ότι παίζει έμμεσο ρόλο στην έναρξη της σκληρωτιογένεσης των μυκήτων S. rolfsii και S. sclerotiorum και άμεσο στων μυκήτων R. solani και S. minor και κύρια πηγή παραγωγής του O2•- είναι τα μιτοχόνδρια και δευτερευόντως η ΧΟ β) η SOD που υπάρχει σε όλους τους μύκητες χρησιμοποιείται από αυτούς για τη μετατροπή του O2•- σε H2O2 γ) η ΧΟ που μέχρι σήμερα δεν ήταν γνωστή η ύπαρξή της στους μύκητες, καθώς και η EC-SOD αλλά και το EC-H2O2, δεν σχετίζονται με τη διαφοροποίηση και ως γνωστόν μπορεί να εμπλέκονται στην προσβολή των φυτών ξενιστών από τους μύκητες αυτούς δ) το H2O2 επάγει την έναρξη της σκληρωτιογένεσης δρώντας σαν παράγοντας σημειακού κυτταρικού πολλαπλασιασμού ε) η CAT που υπάρχει σε όλους τους μύκητες στο αδιαφοροποίητο μόνο στάδιο, μάλλον χρησιμοποιείται από αυτούς ώστε ο κυτταρικός πολλαπλασιασμός να μην γίνεται σε μεγάλη ένταση και αναστείλλει την σημειακή διαφοροποίηση ζ) οι NSPX που εντοπίζονται σε όλους τους μύκητες φαίνεται ότι ελέγχουν τα επίπεδα του H2O2 κυρίως στο διαφοροποιημένο στάδιο η) τα LOOH φαίνεται να δρουν στις μεμβράνες των μυκήτων και σχετίζονται με διαδικασίες μεταβίβασης κυτταρικών μηνυμάτων που επηρεάζουν τον κυτταρικό κύκλο θ) οι μιμητές της SOD Tiron και Tempol, που είχαν ανασταλτική δράση στην διαφοροποίηση θα μπορούσαν να χρησιμοποιηθούν ως μη τοξικά αντιμυκητιακά παρασκευάσματα. / The purpose of this dissertation was the study of sclerotial differentiation, represented by four basic sclerotial types expressed by the filamentous fungi S. rolfsii, R. solani, S. sclerotiorum and S. minor in relation to oxidative stress. Non-sclerotium producing fungi were used as controls of the corresponding wild type strains. The most important peroxides that are responsible for oxidative stress are: a) superoxide radical (O2•-), the primary cause element of oxidative stress, b) hydrogen peroxide (H2O2), produced by the reaction between two O2•- (dismutation reaction), and c) lipid hydroperoxides (LOOH), the primary consequences of oxidative destruction due to free radical attack to polyunsaturated fatty acids. Since there was not available any appropriate method for the quantification of O2•-, a new method was developed for the purpose of this study and its applicability was extended to any organism. In addition, the estimation of the concentration of certain enzymes that regulate the levels of the peroxides mentioned above was also used. These enzymes are a) superoxide dismutase (SOD), which catalyzes the dismutation reaction, b) catalase (CAT), which destroys H2O2, c) non-specific peroxidases (NSPX), which use either H2O2 or LOOH as substrates in order to oxidize other molecules and d) xanthine oxidase (XO), which was detected for the first time in fungi and is responsible, among other functions, for the production of O2•- in organisms. Furthermore, the aldehydic adducts of lipid peroxidation (MDA) were measured in order to evaluate the oxidative destruction of membrane lipids. Finally, specific externally added modulators of the above molecules were used, like: a) SOD mimetics b) H2O2 c) cumene hydroperoxide (lipid hydroperoxide) and d) aminotriazole (CAT inhibitor). The results of this study verified the need for the development of the new method for the quantification of O2•-. Specifically, it was found that the four studied types of sclerotial differentiation are directly related with oxidative stress, and that its components, tested in this study, are formed and changed variously, depending on the type of sclerotial differentiation. The most important conclusions of this study are: a) O2•- plays an indirect role in the initiation of sclerotiogenesis in fungi S. rolfsii and S. sclerotiorum, while in R. solani and S. minor its role is direct, and the main source that produces it in all four fungi are mitochondria and secondarily the ΧΟ enzyme, b) SOD is used by these fungi in order to convert O2•- to H2O2 via the dismutation reaction, c) ΧΟ which was found for the first time in fungi as well as EC-SOD and EC-H2O2, are not related with differentiation and possibly they are involved in tha attack of target plants by these fungi, d) H2O2 induces the initiation of sclerotiogenesis acting as a cell proliferating factor, e) CAT which was found in all fungi only in the undifferentiated stage, is probably used by them in order to control cell proliferation so as to avoid inhibition of sclerotiogenesis, f) NSPX are possibly used by these fungi in order to control H2O2 concentration mainly in the differentiated stage, g) LOOH appear to act on cell membrane and are related to signal transduction processes which affect cell cycle and h) SOD mimetics Tiron and Tempol, which reduced differentiation could be used as non-toxic fungicides.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

Membrane-based separation processes have been used extensively for drinking water purification, wastewater treatment, and numerous other applications. Reactive membranes synthesized through functionalization of the membrane pores offer enhanced reactivity due to increased surface area at the polymer-solution interface and low diffusion limitations. Oxidative techniques utilizing free radicals have proven effective for both the destruction of toxic organics and non-environmental applications. Most previous work focuses on reactions in the homogeneous phase; however, the immobilization of reactants in membrane pores offers several advantages. The use of polyanions immobilized in a membrane or chelates in solution prevents ferric hydroxide precipitation at near-neutral pH, a common limitation of iron(Fe(II/III))-catalyzed hydrogen peroxide (H2O2) decomposition. The objectives of this research are to develop a membrane-based platform for the generation of free radicals, degrade toxic organic compounds using this and similar solution-based reactions, degrade toxic organic compounds in droplet form, quantify hydroxyl radical production in these reactions, and develop kinetic models for both processes. In this study, a functionalized membrane containing poly(acrylic acid) (PAA) was used to immobilize iron ions and conduct free radical reactions by permeating H2O2 through the membrane. The membrane’s responsive behavior to pH and divalent cations was investigated and modeled. The conversion of Fe(II) to Fe(III) in the membrane and its effect on the decomposition of hydrogen peroxide were monitored and used to develop kinetic models for predicting H2O2 decomposition in these systems. The rate of hydroxyl radical production, and hence contaminant degradation can be varied by changing the residence time, H2O2 concentration, and/or iron loading. Using these membrane-immobilized systems, successful removal of toxic organic compounds, such as pentachlorophenol (PCP), from water was demonstrated. Another toxic organic compound of interest for water treatment applications is trichloroethylene (TCE). Due to its limited solubility in water, a majority of the TCE is often present in the form of droplets. In this study, effective TCE droplet degradation using chelate-modified, iron-catalyzed free radical reactions at near-neutral pH was demonstrated. In order to predict the degradation of aqueous and non-aqueous phase TCE for these reactions, a mathematical model was constructed through the use of droplet mass transfer correlations and free radical reaction kinetics.

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H2O2 treatment and compared to that of FeSOD. It’s found that the responses to H2O2 treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe3+ reduction was not observed in Fe(Mn)SOD even H2O2 is believed to oxidize proteins via highly reactive intermediates including Fe and formed via Fe2+, which is true in FeSOD. What’s more, the activities of Fe(Mn)SOD and FeSOD were totally different in the ABTS assay or Amplex Red assay. These results indicated that the mechanism of peroxidase reaction of Fe(Mn)SOD is not identical to that of FeSOD.

The use of lignocellulosic materials to replace fossil resources for the industrial production of fuels, chemicals, and materials is increasing. The carbohydrate composition of lignocellulose (i.e. cellulose and hemicellulose) is an abundant source of sugars. However, due to the feedstock recalcitrance, rigid and compact structure of plant cell walls, access to polysaccharides is hindered and release of fermentable sugars has become a bottle-neck. Thus, to overcome the recalcitrant barriers, thermochemical pretreatment with an acid catalyst is usually employed for the physical or chemical disruption of plant cell wall. After pretreatment, enzymatic hydrolysis is the preferred option to produce sugars that can be further converted into liquid fuels (e.g. ethanol) via fermentation by microbial biocatalysts. However, during acid pretreatment, several inhibitory compounds namely furfural, 5-hydroxymethyl furfural, phenols, and aliphatic acids are released from the lignocellulose components. The presence of these compounds can greatly effect both enzymatic hydrolysis and microbial fermentation. For instance, when Avicel cellulose and acid treated spruce wood hydrolysate were mixed, 63% decrease in the enzymatic hydrolysis efficiency was observed compared to when Avicel was hydrolyzed in aqueous citrate buffer. In addition, the acid hydrolysates were essentially non-fermentable. Therefore, the associated problems of lignocellulose conversion can be addressed either by using feedstocks that are less recalcitrant or by developing efficient pretreatment techniques that do not cause formation of inhibitory byproducts and simultaneously give high sugar yields. A variety of lignocellulose materials including woody substrates (spruce, pine, and birch), agricultural residues (sugarcane bagasse and reed canary grass), bark (pine bark), and transgenic aspens were evaluated for their saccharification potential. Apparently, woody substrates were more recalcitrant than the rest of the species and bark was essentially amorphous. However, the saccharification efficiency of these substrates varied based on the pretreatment method used. For instance, untreated reed canary grass was more recalcitrant than woody materials whereas the acid treated reed canary grass gave a higher sugar yield (64%) than the woody substrates (max 34%). Genetic modification of plants was beneficial, since under similar pretreatment and enzymatic hydrolysis conditions, up to 28% higher sugar production was achieved from the transgenic plants compare to the wild type. As an alternative to the commonly used acid catalysed pretreatments (prior to enzymatic hydrolysis) lignocellulose materials were treated with four ionic liquid solvents (ILs): two switchable ILs (SILs) -SO2DBUMEASIL and CO2DBUMEASIL, and two other ILs [Amim][HCO2] and [AMMorp][OAc]. viii After enzymatic hydrolysis of IL treated substrates, a maximum amount of glucan to glucose conversion of between 75% and 97% and a maximum total sugar yields of between 71% and 94% were obtained. When using acid pretreatment these values varied between 13-77% for glucan to glucose conversion and 26-83% for total sugar yield. For woody substrates, the hemicellulose recovery (max 92%) was higher for the IL treated substrates than compared to acid treated samples. However, in case of reed canary grass and pine bark the hemicellulose recovery (90% and 88%, respectively) was significantly higher for the acid treated substrates than the IL treated samples. To overcome the inhibitory problems associated with the lignocellulose hydrolysates, three chemical conditioning methods were used 1. detoxification with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2) 2. application of reducing agents (sulfite, dithionite, or dithiothreitol) and 3. treatment with alkali: Ca(OH)2, NaOH, and NH4OH. The concentrations of inhibitory compounds were significantly lower after treatments with FeSO4 and H2O2 or alkali. Using reducing agents did not cause any decrease in the concentration of inhibitors, but detoxification of spruce acid hydrolysates resulted in up to 54% improvement of the hydrolysis efficiency (in terms of sugar release) compared to untreated samples. On the other hand, application of detoxification procedures to the aqueous buffer resulted in up to 39% decrease in hydrolysis efficiency, thus confirming that the positive effect of detoxification was due to the chemical alteration of inhibitory compounds. In addition, the fermentability of detoxified hydrolysates were investigated using the yeast Saccharomyces cerevisiae. The detoxified hydrolysates were readily fermented to ethanol yielding a maximum ethanol concentration of 8.3 g/l while the undetoxified hydrolysates were basically non-fermentable.

The study aimed to investigate the following: 1. Investigate a putative glutathione peroxidase gene (Glyma17g34110) within Glycine max by an in silico analysis and spatial expression. 2. Determine the effects of exogenously applied nitric oxide on the expression of Glyma17g34110. 3. Investigate the antioxidant mechanism with attention to Glyma17g34110,reactive oxygen species and cell death in the response to salt stress. 4. Establish whether Glyma17g34110 is a glutathione peroxidase or thioredoxindependent peroxidase gene. / Magister Scientiae - MSc

The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress. / >Magister Scientiae - MSc

There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1¿M and 10¿M induced increases of over ~10-fold in the percentage of apoptotic cells (p¿0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.