Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

Visible light inactivation of bacteria and fungi by modified titanium dioxide.

Visible light induced photocatalytic inactivation of bacteria (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis) and fungi (Candida albicans, Aspergillus niger) was tested. Carbon-doped titanium dioxide and TiO2 modified with platinum(IV) chloride complexes were used as suspension or immobilised at the surface of plastic plates. A biocidal effect was observed under visible light irradiation in the case of E. coli in the presence of both photocatalysts. The platinum(IV) modified titania exhibited a higher inactivation effect, also in the absence of light. The mechanism of visible light induced photoinactivation is briefly discussed. The observed detrimental effect of photocatalysts on various microorganism groups decreases in the order: E. coli > S. aureus approximately E. faecalis>C. albicans approximately A. niger. This sequence results most probably from differences in cell wall or cell membrane structures in these microorganisms and is not related to the ability of catalase production.     

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qβ and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.     

In vitro inactivation of methionine synthase by nitrous oxide

Nitrous oxide (N2O) is commonly used as an anesthetic agent. Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates. We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O. Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system. Reisolation of the inactivated E. coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation. However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin. We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical. Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity.     

核壳结构CdS-TiO_2纳米颗粒体外光催化灭活白血病HL60细胞实验研究

本文研究了核壳结构CdS-TiO_2纳米颗粒光对HL60细胞光催化灭活作用,并初步探讨了CdS量子点增强TiO_2光催化灭活效率的作用机理。实验利用超声法制备了核壳结构CdS-TiO_2纳米颗粒,使用CCK-8法检测了其对白血病HL60细胞的光催化灭活效果,并采用活性氧分析和荧光发光光谱等分析手段对CdS量子点增强TiO_2光催化灭活效率的作用机理进行了分析。细胞实验结果表明,在暗室条件下,随着CdS包裹的TiO_2外壳的增厚,细胞的暗室存活率从28.5%逐渐上升至80%;在可见光照下,细胞的存活率显著下降。CdS-TiO_2纳米颗粒对HL60细胞的光催化灭活率均超过60%,其中CdS-TiO_2样品0.6对HL60细胞的光催化灭活效率最高,达到95%。根据荧光光谱和活性氧含量分析的结果推测,这可能是由于核壳结构的CdS内核与TiO_2外壳之间产生了有效的电子转移,抑制了TiO_2的空穴电子的复合,增强了TiO_2外壳的光催化活性,最终增加了TiO_2对HL60细胞的PDT灭活效果。     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

The role of radical reactions in organomercurials impact on lipid peroxidation

The organomercury compounds RHgX and R(2)Hg are broad-spectrum biocidal agents acting via diverse mechanisms in biological systems. Despite the enormous amount of studies carried out in last decades to elucidate the detailed mechanisms of organomercurials toxicity their biomolecular mode of action is still under debate. Among various toxicity mechanisms the action of RHgX and R(2)Hg at the membrane level due to the lipophilic properties of their molecules is discussed. Organomercurials are supposed to induce membrane associated oxidative stress in living organism through different mechanisms including the enhancement of the lipid peroxidation and intracellular generation of reactive oxygen species (ROS), H(2)O(2), O(2)(-), HO(). The perturbation of antioxidative defense system and the peroxidation of unsaturated fatty acids in membrane lipid bilayer are consequences of this impact. On the other hand, the involvement of organomercurials in radical and redox biochemical processes is manifested in carbon to metal bond cleavage that leads to the generation of reactive organic radicals R(). This pathway is discussed as one of the multiple mechanisms of organomercurials toxicity. The goal of this review is to present recent results in the studies oriented towards the role of organomercurials in the xenobiotic-mediated enhancement of radical production and hence in the promotion of lipids peroxidation. The application of natural and synthetic antioxidants as detoxification agents is presented.     

Effect of the photocatalytic activity of TiO(2) on plasmid DNA

We investigated the photodynamic DNA strand-breaking activity of TiO(2). A solution of super-coiled pBR 322 DNA was irradiated with 5 J/cm(2) of UVA in the presence of TiO(2) and the products were analyzed by using gel electrophoresis. The ratio of open-circular DNA to super-coiled circular DNA was calculated from the resulting peak areas as a DNA strand-breaking index (SBI). The SBI of anatase-structure TiO(2) (band gap=3.23 eV) was greater than that of rutile structure (band gap=3.06 eV), and the level of SBI correlated with the photocatalytic activity for degradation of 2-propanol. The inhibitory effects of active oxygen scavengers, including DMSO, glutathione and histidine, on the DNA strand-breaking activity were examined. All of the scavengers except ascorbic acid showed inhibitory effects, as did several polyhydric alcohols including mannitol, a well-known hydroxyl radical scavenger. These results suggest that the photodynamic DNA strand-breaking activity of TiO(2) is due to active oxygen species, especially hydroxyl radicals. Polyhydric alcohols showed an inverse correlation between the inhibitory effect on DNA strand-breaking activity and the octanol/water partition coefficient (logP).     

Killing of bacillus spores by aqueous dissolved oxygen,ascorbic acid,and copper ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.     

Differential contractile actions of reactive oxygen species on rat aorta: selective activation of ATP receptor by H2O2

This study aims to examine the effects of different reactive oxygen species (ROS) on the resting tension of endothelium-denuded rat aortic rings. In these preparations, H2O2 (30 microM) induced a fast and transient contraction, which could be abolished by pretreatment of catalase (800 U/ml), but not affected by superoxide anion scavenger, superoxide dismutase (SOD; 150 U/ml) or the hydroxyl free radical scavenger, DMSO/mannitol (each 3 mM). In contrast, pyrogallol, a putative superoxide anion donor, induced a biphasic contraction, which could be abolished by SOD, but not by catalase or DMSO/mannitol. Unlike H2O2 and pyrogallol, Vitamin C(VitC)/Fe2+ (each 100 microM), a commonly used hydroxyl radical-generating system, triggered a tonic contraction which could be prevented by DMSO/mannitol, but not by SOD or catalase. Interestingly, H2O2-induced contraction could be concentration-dependently (10-100 microM) inhibited by suramin and reactive blue-2 (RB-2), two widely used ATP receptor antagonists. On the other hand, suramin or RB-2, at concentration up to 100 microM, affected neither pyrogallol nor VitC/Fe2+-induced contraction. In conclusion, we showed for the first time that different ROS could contract rat aorta with different mechanisms of action, and H2O2 elicits a transient contraction probably as a result of the ATP receptor activation.     

A non-hypoxic, ROS-sensitive pathway mediates TNF-alpha-dependent regulation of HIF-1alpha

A non-hypoxic, reactive oxygen species (ROS)-sensitive pathway mediating tumor necrosis factor-alpha (TNF-alpha)-dependent regulation of hypoxia-inducible factor-1alpha (HIF-alpha) was investigated in vitro. TNF-alpha mediated the translocation of HIF-1alpha, associated with up-regulating its activity under normoxia. Analysis of the mode of action of TNF-alpha revealed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O(2-.)) and hydroxyl radical (.OH). Antioxidants purported as prototypical scavengers of H2O2 and .OH, attenuated TNF-alpha-induced HIF-1alpha activation, and blockading NADPH-oxidase by scavenging O(2-.) reduced the activity of HIF-1alpha. Inhibition of the mitochondrion complex I abrogated TNF-alpha-dependent activation of HIF-1alpha. Interrupting the respiratory chain reversed the excitatory effect of TNF-alpha on HIF-1alpha. These results indicate a non-hypoxic pathway mediating cytokine-dependent regulation of HIF-1alpha in a ROS-sensitive mechanism.     

The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide

The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure. Two modes of killing were detected following a 15-min challenge with H2O2 under either aerated or anoxic conditions. Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM. Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic cells. Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H2O2. Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations. We suggest that hydroxyl radicals mediate mode two but not mode one killing by H2O2.     

Salicylic acid-induced inactivation of creatine kinase in the presence of lactoperoxidase and H2O2

To clarify one mechanism of aspirin-induced gastric mucosal damage, inactivation of creatine kinase (CK) by salicylic acid that is easily produced from aspirin in vivo was examined in the presence of lactoperoxidase (LPO) and H2O2 (LPO-H2O2). Salicylic acid inactivated CK (rabbit muscle) during its interaction with LPO-H2O2. CK activity in gastric mucosal homogenate decreased dependent on the concentration of salicylic acid in the presence of LPO-H2O2. Oxygen radical scavengers did not prevent the inactivation of CK. Direct detection of free radicals of salicylic acid by electron spin resonance was unsuccessful. However, glutathionyl radicals were formed during the interaction of salicylic acid with LPO-H2O2 in the presence of reduced glutathione and 5,5-dimethyl-1-pyrroline oxide as a spin trap agent. Among salicylic acid-related drugs, salsalate, but not aspirin and ethenzamide, inactivated CK, indicating the phenolic hydroxyl group is oxidized by LPO-H2O2. During oxidation of salicylic acid by LPO-H2O2, the sulfhydryl group in CK markedly decreased, and salicylic acid bound to CK. These results indicate that CK was inactivated through loss of the sulfhydryl group and binding of salicylic acid.     

Antiinflammatory drugs: protection of a bacterial virus as an in vitro biological measure of free radical activity

The effects of the hydroxyl free radical (OH), the superoxide free radical (O2-) and the trichloromethyl peroxy free radical (CC13O2) on the survival of bacteriophage T2 have been studied in the absence and presence of several non-steroidal anti-inflammatory drugs (NSAID). The trichloromethylperoxy radical derived from carbon tetrachloride is considerably more effective than the hydroxyl radical in inactivating the virus: the superoxide radical has only a minor inactivating effect. All the NSAID investigated (flurbiprofen, ibuprofen, sulindac, piroxicam, benoxaprofen, mefenamic acid, diflunisal, aspirin, D-penicillamine, indomethacin and metiazinic acid) inhibit inactivation by OH. This is in agreement with the high rate constants of reaction with this radical determined using the fast reaction technique of pulse radiolysis, i.e. (k greater than 10(9) M-1 S-1). The sulphur-containing drugs, metiazinic acid, piroxicam, penicillamine and sulindac as well as the indole derivative indomethacin, protect the virus from inactivation by the model peroxy radical CC13O2 (the dose modifying factor, DMF greater than 20). In contrast, acetylsalicylic acid related drugs, such as diflunisal, the anthranilic acid derivative, mefenamic acid, and some phenylpropionic acid derivatives, such as flurbiprofen, exhibit only a very small or no protective effect (DMF less than 2). As with OH, the ability of the drugs to protect the virus from inactivation by the peroxy radical is in agreement with their corresponding rate constants of reaction determined by pulse radiolysis.     

Plasma membrane-generated reactive oxygen intermediates and their role in cell growth of plants

Reactive oxygen species (ROS) produced as intermediates in the reduction of O2 to H2O (superoxide radical, hydrogen peroxide, hydroxyl radical), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry can also play useful roles if it takes place outside of the protoplast. In plants, the production of these ROS initiated by the plasma membrane NAD(P)H oxidase can be used for controlled polymer breakdown leading to wall loosening during extension growth. Backbone cleavage of cell wall polysaccharides can be accomplished by hydroxyl radicals produced from hydrogen peroxide and superoxide in a reaction catalyzed by cell wall peroxidase. Growing plant organs such as coleoptiles or roots of maize seedlings produce these ROS specifically in the apoplast of actively growing tissues, e.g. in the epidermis of the coleoptile and the growing zone of the root. Auxin promotes the release of hydroxyl radicals when inducing elongation growth. Experimental generation of hydroxyl radicals in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ROS or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which the generation of hydroxyl radicals, initiated by the plasma membrane NAD(P)H oxidase, plays a central role.     

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Reaction of beta-alkannin (shikonin) with reactive oxygen species: detection of beta-alkannin free radicals

beta-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of beta-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of beta-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen ((1)O2). superoxide anion radical (.O2), hydroxyl radical (.OH) and tert-butyl peroxyl radical (BuOO.) as well as iron-dependent microsomal lipid peroxidation. During the reactions of beta-alkannin with 1O2, .O2- and BuOO., intermediate organic radicals due to beta-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of beta-alkannin, the beta-alkannin radical observed as an intermediate in the reactions with (1)O2, and .O2- was concluded to be a semiquinone radical. On the other hand, during the reactions of beta-alkannin and naphthazarin with BuOO., ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of beta-alkannin by BuOO.. Based on the ROS-scavenging abilities of beta-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.     

Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism

Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.