Potential of unglycosylated horseradish peroxidase variants for enzyme prodrug cancer therapy.

Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA). However, the plant-derived enzyme is out of bounds for medical purposes due to its foreign glycosylation pattern and resulting rapid clearance and immunogenicity. In this study, we generated recombinant, unglycosylated HRP variants in Escherichia coli using random mutagenesis and investigated their biochemical properties and suitability for cancer treatment. The cytotoxicity of the HRP-IAA enzyme prodrug system was assessed in vitro with HCT-116 human colon, FaDu human nasopharyngeal squamous cell carcinoma and murine colon adenocarcinoma cells (MC38). Extensive cytotoxicity was shown in all three cancer cell lines: the cell viability of HCT-116 and MC38 cells treated with HRP-IAA was below 1% after 24 h incubation and the surviving fraction of FaDu cells was ≤ 10% after 72 h. However, no cytotoxic effect was observed upon in vivo intratumoral application of HRP-IAA on a MC38 tumor model in C57BL/6J mice. However, we expect that targeting of HRP to the tumor by conjugation to specific antibodies or antibody fragments will reduce HRP clearance and thereby enhance therapy efficacy.

Control of cellular adhesiveness in hyaluronic acid-based hydrogel through varying degrees of phenol moiety cross-linking.

Current hyaluronic acid-based hydrogels often cause cytotoxicity to encapsulated cells and lack the adhesive property required for effective biomedical and tissue engineering applications. Provision of the cell-adhesive surface is an important requirement to improve its biocompatibility. An aqueous solution of hyaluronic acid possessing phenolic hydroxyl (HA-Ph) moieties is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative cross-linking reaction. This study evaluates the effect of different degrees of cross-linked Ph moieties on cellular adhesiveness and proliferation on the resultant enzymatically cross-linked HA-Ph hydrogels. Mechanical characterization demonstrated that the compression force of engineered hydrogels could be tuned in the range of 0.05-35 N by changing conjugated Ph moieties in the precursor formulation. The water contact angle and water content show hydrophobicity of hydrogels increased with increasing content of cross-linked Ph groups. The seeded mouse embryo fibroblast-like cell line and human cervical cancer cell line, on the HA-Ph hydrogel, proved cell attachment and spreading with a high content of cross-linked Ph groups. The HA-Ph with a higher degree of Ph moieties shows the maximum degree of cell adhesion, spreading, and proliferation which presents this hydrogel as a suitable biomaterial for biomedical and tissue engineering applications.

Evaluation of colorimetric assays for determination of H2O2in planta during fungal wood decomposition.

Hydrogen peroxide (H2O2) plays a critical role in generating oxygen radicals as fungi attack and deconstruct plant cell walls. Its concentrations in planta, however, are often low during decomposition and evade detection by traditional methods. Here, we compared relevant methods and selected the best based on detection limits and selectivity.

Recombinant horseradish peroxidase variants for targeted cancer treatment.

Cancer is a major cause of death. Common chemo- and radiation-therapies damage healthy tissue and cause painful side effects. The enzyme horseradish peroxidase (HRP) has been shown to activate the plant hormone indole-3-acetic acid (IAA) to a powerful anticancer agent in in vitro studies, but gene directed enzyme prodrug therapy (GDEPT) studies showed ambivalent results. Thus, HRP/IAA in antibody directed enzyme prodrug therapy (ADEPT) was investigated as an alternative. However, this approach has not been intensively studied, since the enzyme preparation from plant describes an undefined mixture of isoenzymes with a heterogenic glycosylation pattern incompatible with the human system. Here, we describe the recombinant production of the two HRP isoenzymes C1A and A2A in a Pichia pastoris benchmark strain and a glyco-engineered strain with a knockout of the α-1,6-mannosyltransferase (OCH1) responsible for hypermannosylation. We biochemically characterized the enzyme variants, tested them with IAA and applied them on cancer cells. In the absence of H2 O2 , HRP C1A turned out to be highly active with IAA, independent of its surface glycosylation. Subsequent in vitro cytotoxicity studies with human T24 bladder carcinoma and MDA-MB-231 breast carcinoma cells underlined the applicability of recombinant HRP C1A with reduced surface glycoslyation for targeted cancer treatment. Summarizing, this is the first study describing the successful use of recombinantly produced HRP for targeted cancer treatment. Our findings might pave the way for an increased use of the powerful isoenzyme HRP C1A in cancer research in the future.

Feedback from visual cortical area 7 to areas 17 and 18 in cats: How neural web is woven during feedback.

To investigate the feedback effect from area 7 to areas 17 and 18, intrinsic signal optical imaging combined with pharmacological, morphological methods and functional magnetic resonance imaging (fMRI) was employed. A spatial frequency-dependent decrease in response amplitude of orientation maps was observed in areas 17 and 18 when area 7 was inactivated by a local injection of GABA, or by a lesion induced by liquid nitrogen freezing. The pattern of orientation maps of areas 17 and 18 after the inactivation of area 7, if they were not totally blurred, paralleled the normal one. In morphological experiments, after one point at the shallow layers within the center of the cat's orientation column of area 17 was injected electrophoretically with HRP (horseradish peroxidase), three sequential patches in layers 1, 2 and 3 of area 7 were observed. Employing fMRI it was found that area 7 feedbacks mainly to areas 17 and 18 on ipsilateral hemisphere. Therefore, our conclusions are: (1) feedback from area 7 to areas 17 and 18 is spatial frequency modulated; (2) feedback from area 7 to areas 17 and 18 occurs mainly ipsilaterally; (3) histological feedback pattern from area 7 to area 17 is weblike.

A glucose-powered antimicrobial system using organic-inorganic assembled network materials.

A new glucose-driven photodynamic antimicrobial system was developed to efficiently kill bacteria and fungi, taking advantage of organic-inorganic network materials encapsulating glucose oxidase and horseradish peroxidase and bioluminescence resonance energy transfer (BRET).

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies.

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis.

Indole-3-acetic acid/horseradish peroxidase induces apoptosis in TCCSUP human urinary bladder carcinoma cells.

Indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) have emerged as a new strategy for cancer treatment. In the present study, we determined the effects of IAA/HRP treatment on TCCSUP human urinary bladder carcinoma cells. It was found that the IAA/HRP combination decreased cell viability of TCCSUP cells in a time- and dose-dependent manner, whereas IAA or HRP alone showed no such effect. In addition, the decreased cell viability was restored by pretreatment with ascorbic acid. To clarify the mechanism of death of TCCSUP cells by IAA/HRP, we investigated the signal transduction pathways related to the apoptosis. It was found that IAA/HRP activates p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We further investigated the IAA/HRP-mediated apoptotic pathways and showed that IAA/HRP induces caspase-8 and caspase-9 activation, which results in caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. To further confirm whether IAA/HRP induces apoptotic cell death, we performed a DNA fragmentation assay after IAA/HRP treatment and found that IAA/HRP-treated cells showed typical apoptotic DNA ladder formation. From these results, we suggest that IAA/HRP induces apoptosis of TCCSUP human urinary bladder carcinoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Bioactivation of minocycline to reactive intermediates by myeloperoxidase, horseradish peroxidase, and hepatic microsomes: implications for minocycline-induced lupus and hepatitis.

Of the tetracyclines, minocycline is unique in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. It is also unique among the tetracyclines in having a para-N,N-dimethylaminophenol ring. Many drugs that cause autoimmune reactions are oxidized to reactive metabolites by the myeloperoxidase (MPO) system of macrophages. In this study, we showed that minocycline is oxidized to reactive intermediates by MPO/H(2)O(2)/Cl(-), HOCl, horseradish peroxidase/H(2)O(2), or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N,N-dimethylamino group, which implies that the reactive intermediate was a quinone iminium ion. The NMR of the other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings. In the oxidation by HOCl, we found an intermediate with a protonated molecular ion of m/z 510 that represents the addition of HOCl to minocycline. The HOCl presumably adds across the double bond of the B ring, and reaction of this intermediate with NAC led to the second NAC adduct. We were surprised to find that the same NAC adduct was not observed after oxidation of tetracycline with HOCl, even though this part of the tetracycline structure is the same as for minocycline. We propose that one or more of these reactive metabolites are responsible for the idiosyncratic drug reactions that are specific to this tetracycline.

A solid-in-oil nanodispersion for transcutaneous protein delivery.

Transcutaneous delivery attracts much attention but remains a challenging strategy for hydrophilic macromolecular drug administration. In the present study, we demonstrated that a solid-in-oil (S/O) nanodispersion, an oil-based nanodispersion of hydrophilic drugs, effectively enhanced the permeation of proteins into the skin. All of the different model proteins, FITC-labeled insulin (MW ca. 6 kDa), enhanced green fluorescent protein (EGFP, MW ca. 27 kDa) and horseradish peroxidase (HRP, MW ca. 40 kDa), permeated through the stratum corneum of Yucatan micropig skin in vitro by forming a S/O nanodispersion. The penetrated EGFP and HRP exhibited green fluorescence and catalytic activity, respectively, suggesting that these proteins can permeate into the skin in a functional form. The results indicated the potential utility of the S/O nanodispersion as a novel vehicle for transcutaneous protein delivery.

Effect of reactive oxygen intermediaries on the viability and infectivity of Mycobacterium lepraemurium.

Murine leprosy is a natural disease of the mouse, the most popular model animal used in biomedical research; the disease is caused by Mycobacterium lepraemurium (MLM), a successful parasite of macrophages. The aim of the study was to test the hypothesis that MLM survives within macrophages because it highly resists the toxic effects of the reactive oxygen intermediaries produced by these cells in response to infection by the microorganism. MLM cells were incubated in the presence of horseradish peroxidase (HRPO)-H(2)O(2)-halide for several periods of time. The peroxidative effect of this system was investigated by assessing the changes occurred in (a) lipid composition; (b) viability; and (c) infectivity of the microorganism. Changes in the lipid composition of peroxidated- vs. intact-MLM were analysed by thin layer chromatography. The effect of the peroxidative system on the viability and infectivity of MLM was measured by the alamar blue reduction assay and by its ability to produce an infection in the mouse, respectively. Peroxidation of MLM produced drastic changes in the lipid envelope of the microorganism, killed the bacteria and abolished their ability to produce an in vivo infection in the mouse. In vitro, MLM is highly susceptible to the noxious effects of the HRPO-H(2)O(2)-halide system. Although the lipid envelope of MLM might protect the microorganism from the peroxidative substances produced at 'physiological' concentrations in vivo, the success of MLM as a parasite of macrophages might rather obey for other reasons. The ability of MLM to enter macrophages without triggering these cells' oxidative response and the lack of granular MPO in mature macrophages might better explain its success as an intracellular parasite of these cells.

Comparative cytotoxicity and ROS generation by curcumin and tetrahydrocurcumin following visible-light irradiation or treatment with horseradish peroxidase.

In order to clarify the cytotoxic mechanism of curcumin, a well-known chemopreventive agent, the cytotoxicity (by MTT method), intracellular glutathione (using GSH detection kit) and intracellular reactive oxygen species (ROS) levels (with a flow cytometer), were measured in curcumin- and tetrahydrocurcumin (TH-curcumin)-treated cancer (HSG) and normal (HGF) cells under two different oxidation conditions: irradiation with visible light (VL) and enzymatic oxidation with horseradish peroxidase (HRP)/H2O2. The cytotoxicity of curcumin was highly enhanced by VL-irradiation, whereas that of TH-curcumin was enhanced by HRP/H2O2 treatment. The cytotoxicity of curcumin against HGF cells was greater than that against HSG cells. Curcumin significantly reduced the intracellular GSH level significantly under VL-irradiation, and increased it under HRP/H2O2, whereas TH-curcumin had no effect with either oxidation treatment. HRP/H2O2 treatment of TH-curcumin enhanced generation of ROS; in contrast, VL-irradiation of curcumin was considered to produce ROS preferably. In conclusion, curcumin was highly photo-toxic, caused a decrease in GSH and mediated ROS generation. In contrast, the cytotoxicity of TH-curcumin was enhanced by enzymatic oxidation. A low-level pro-oxidant intracellular milieu induced by TH-curcumin could be effectively useful for cancer prevention.

Indole-3-acetic acid/horseradish peroxidase-induced apoptosis involves cell surface CD95 (Fas/APO-1) expression.

Recently, we showed that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) produces hydrogen peroxide (H2O2), and that this leads to the apoptosis of G361 human melanoma cells. In the present study, flow cytometric analysis confirmed that H2O2 is involved the IAA/HRP-induced apoptotic process. We also found that IAA/HRP increases cell surface CD95 (Fas/APO-1) expression, and that this is blocked by catalase treatment. Furthermore, blocking CD95 with a neutralizing antibody significantly restored IAA/HRP-induced apoptosis. In addition, the IAA/HRP-induced activations of CD95 downstream molecules, i.e., caspase-8, Bid, and caspase-3, were also inhibited by catalase. Moreover, a caspase-8 inhibitor significantly blocked IAA/HRP-induced apoptosis. These results indicate that IAA/HRP-induced apoptosis involves a CD95-initiated death receptor signaling pathway initiated by hydrogen peroxide.

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis.

Recently, we reported that a combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP-induced apoptosis. Our results show that IAA/HRP-induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP-induced apoptotic cell death, indicating that IAA/HRP-produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP-mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP-induced apoptosis.

Vimang (Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component.

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 microM Ca(2+), induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca(2+) release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H(2)O(2)/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.

[Mechanism of apoptosis induced by indole-3-acetic acids combined with horseradish peroxidase in leukemia cell line K562].

To investigate the possible mechanism of apoptosis induced by indole-3-acetic acid (IAA) combined with horseradish peroxidase in leukemia cell line K562, cell proliferation and apoptosis of K562 cell were examined by MTT assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively; the activity of superoxide dismutase (SOD) and the quantitative change of MDA were measured by biochemical method; changes of free radical were determined by 2, 7-dichlorofluorescin diacetate (DCFH-DA) probe with confocal microscopy. The results showed that of MTT assay and TUNEL indicated that IAA/HRP could significantly inhibit cell proliferation (P < 0.05) and induce apoptosis of K562 cell (P < 0.01), at the same time a positive correlation was found between apoptosis rate and IAA concentration (r = 0.971, P < 0.01). The activity of SOD and the quantitative of MDA increased, accompanied with a rise in IAA concentration. Results detected by DCFH-DA probe indicated that the fluorescence intensity of intracellular free radical increased, as compared with control, and a positive correlation was found. It is concluded that IAA/HRP can inhibit proliferation of K562 cells and induce K562 cell apoptosis, its mechanism may be related with the increase of intracellular free radical due to the effects of IAA/HRP.

Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination with horseradish peroxidase.

AIM: To explore the mechanisms underlying the apoptosis of human pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid (IAA) in combination with horseradish peroxidase (HRP). METHODS: BXPC-3 cells derived from human pancreatic cancer were exposed to 40 or 80 micromol/L IAA and 1.2 microg/mL HRP at different times. Then, MTT assay was used to detect the cell proliferation. Flow cytometry was performed to analyze cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to detect apoptosis. 2,7-Dichlorofluorescin diacetate uptake was measured by confocal microscopy to determine free radicals. Level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. RESULTS: IAA/HRP initiated growth inhibition of BXPC-3 cells in a dose- and time-dependent manner. Flow cytometry revealed that the cells treated for 48 h were arrested at G1/G0. After exposure to 80 micromol/L IAA plus 1.2 microg/mL HRP for 72 h, the apoptosis rate increased to 72.5 per thousand, which was nine times that of control. Content of MDA and activity of SOD increased respectively after treatment compared to control. Meanwhile, IAA/HRP stimulated the formation of free radicals. CONCLUSION: The combination of IAA and HRP can inhibit the growth of human pancreatic cancer BXPC-3 cells in vitro by inducing apoptosis.

Intestinal growth and differentiation in zebrafish.

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

Potent antioxidative and antigenotoxic activity in aqueous extract of Japanese rice bran--association with peroxidase activity.

To estimate the preventive potential of Japanese rice bran (Oryza sativa japonica) against the oxygen radical-related chronic diseases such as cardio-vascular diseases and cancer, antioxidative and antigenotoxic activities of the rice bran extracts were analyzed by using assay systems for lipid peroxidation and genotoxin-induced umu gene expression. When effects of the rice bran extracts under different extraction conditions on hydroperoxide generation from auto-oxidized linoleic acid were examined using aluminum chloride method, the water extract showed strong antioxidant activity, but the methanol and acetone extracts did not exhibit significant activity. The water extract of rice bran was divided into the ethanol-precipitable (EP) and supernatant fractions, and EP fraction showed the dominant antioxidant activity, but the supernatant fraction did not exhibit significant antioxidant activity. When the effect of EP fraction on umu C gene expression in SOS response associated with DNA damage in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was analyzed, it showed a dose-dependent suppressive activity against Trp-P-1-induced umu C gene expression. The bio-chemical analysis of EP fraction indicates that the major antioxidative and antigenotoxic activity of EP fraction is associated with a proteinous component with the molecular weight of more than 30 KDa. As a possible active principle for the antioxidative and antigenotoxic activity in EP fraction, the strong activity of an oxygen radical-scavenging enzyme, peroxidase was detected, and the purified horseradish peroxidase also caused the similar antioxidative and antigenotoxic activities. The significance of this finding is discussed from the viewpoint of the preventive role of rice bran against oxygen radical-related chronic diseases.

Prooxidant activity and cytotoxic effects of indole-3-acetic acid derivative radicals.

Previously, it was shown that indole-3-acetic acid (IAA) is a nontoxic prodrug that forms a radical, toxic to tumor cells when activated by peroxidase. Because of this, IAA and peroxidase conjugated to an antibody specific to an extracelluar tumor antigen are currently in phase II clinical trials. In the following, the prooxidant activities of the radicals formed were compared when IAA or its derivatives were metabolically oxidized by peroxidase/H(2)O(2). In general, it was found that the effectiveness of IAA analogues for catalyzing the cooxidation of ascorbate, NADH, or GSH increased as the IAA derivatives were more readily oxidized by HRP/H(2)O(2). The order of effectiveness of IAA derivatives at cooxidizing NADH, ascorbate, GSH, and hepatocyte GSH was 5MeO-2Me-IAA > 2Me-IAA > 5MeO-IAA > IAA. The rates of NADH and ascorbate cooxidation were faster at pH 7.4 than at pH 6.0, whereas GSH cooxidation was faster at pH 6.0 than at pH 7.4. Furthermore, NADH, ascorbate, and GSH prevented the oxidation of IAA derivatives, which suggested that the indolyl cation radical was responsible for the prooxidant activity. The effectiveness of IAA derivatives in catalyzing lipid peroxidation at pH 7.4 was similar and also correlated with the rate of oxidation of IAA derivatives by HRP-I and the one-electron potential of these compounds. The IAA derivative-induced lipid peroxidation was faster at pH 6.0 than at pH 7.4. IAA derivative effectiveness at catalyzing microsomal and hepatocyte lipid peroxidation or hepatocyte reactive oxygen species formation at pH 6.0 was IAA > 5MeO-2Me-IAA > 2Me-IAA > 5MeO-IAA, but at pH 7.4, it was 5MeO-2Me-IAA > 2Me-IAA > 5MeO-IAA > IAA. Previously, the rate of radical cation decarboxylation to skatole radicals and (skatole) peroxyl radicals was reported to be faster at an acid pH with IAA being more effective than the derivatives. This suggests that IAA skatole and/or (skatole) peroxyl radicals catalyze lipid peroxidation at pH 6.0. Incubation of isolated rat hepatocytes with IAA analogues/H(2)O(2)/peroxidase also resulted in cytotoxicity with 5MeO-2Me-IAA being the most effective at pH 7.4 and IAA being the most effective at pH 6.0. Cytotoxicity was also prevented by antioxidants.