Bioinspired Microenvironment Responsive Nanoprodrug as an Efficient Hydrophobic Drug Self-Delivery System for Cancer Therapy.

Artemisinin compounds have shown satisfactory safety records in anti-malarial clinical practice over decades and have revealed value as inexpensive anti-tumor adjuvant chemotherapeutic drugs. However, the rational design and precise preparation of nanomedicines based on the artemisinin drugs are still limited due to their non-aromatic and fragile chemical structure. Herein, a bioinspired coordination-driven self-assembly strategy was developed to manufacture the artemisinin-based nanoprodrug with a significantly increased drug loading efficacy (∼70 wt %) and decreased preparation complexity compared to conventional nanodrugs. The nanoprodrug has suitable size distribution and robust colloidal stability for cancer targeting in vivo. The nanoprodrug was able to quickly disassemble in the tumor microenvironment with weak acidity and a high glutathione concentration, which guarantees a better tumor inhibitory effect than direct administration and fewer side effects on normal tissues in vivo. This work highlights a new strategy to harness a robust, simplified, organic solvent-free, and highly repeatable route for nanoprodrug manufacturing, which may offer opportunities to develop cost-effective, safe, and clinically available nanomedicines.

Hyaluronic acid-based nanogels derived from multicomponent self-assembly for imaging-guided chemo-photodynamic cancer therapy.

Multifunctional theranostic nanoplatforms integrated of imaging function, multi-modality therapy, stimuli-responsiveness, and targeted delivery are of highly desirable attributes in achieving precise medicine. However, preparation of multifunctional nanoplatforms often involves laborious, multiple steps and inevitably utilizes low-biocompatible or non-functional components. Herein we report a facile, one-step self-assembly strategy to fabricate hyaluronic acid (HA)-based multifunctional tumor theranostic nanoplatform by employing magnetic resonance imaging (MRI) agent Mn2+ as a reversible crosslink agent for histidine-grafted HA, along with simultaneously loading chemotherapeutic agent doxorubicin hydrochloride (DOX) and photodynamic therapy agent chlorin e6, to realize MRI-guided targeted chemo-photodynamic cancer therapy. The targeted delivery and stimuli-responsive payload release were demonstrated in vitro and in vivo. Furthermore, the combined chemo-photodynamic therapy of the nanoassembly dramatically improved the cancer therapeutic outcome, in comparison with that of free DOX and nanoplatform solely loaded DOX in a melanoma bearing mice. Our one step assemble strategy is of great potential in clinic transformation.

HSP70 induction by bleomycin metal core analogs.

Induction of heat shock protein 70 (HSP70) is known to be effective against various diseases. We are interested in HSP70 induction capability of an antitumor antibiotic bleomycin which produces oxidative stress by iron chelate formation and oxygen activation in a cell. The HSP70 induction activity of bleomycin and its six metal core analogs was examined, and a compound HPH-1Trt of 10 µM was found to induce this protein in a pheochromocytoma cell line and some T cell and monocytic cell lines. Its mechanism is increase of HSP70 mRNA, but higher concentration of this compound showed toxicity. Two new derivatives were then synthesized, and one of them named DHPH-1Trt was shown to have less toxicity and higher HSP70 induction activity. This study would lead to a clue for new HSP70 inducer clinically used in near future.

Cyclosporine A as a Cardioprotective Agent During Donor Heart Retrieval, Storage, or Transportation: Benefits and Limitations.

BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage. METHODS: After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.2 µM), then reperfused for 45 minutes in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior-alone stored hearts were exposed to 0.2 µM CsA for the initial 15 minutes (nonworking period) or the full 45-minute period of reperfusion. Coronary effluent was collected pre- and poststorage for assessment of lactate dehydrogenase release. Tissue samples were collected at the end of each study for immunoblotting and histological studies. RESULTS: CsA supplementation during cold storage or the first 15-minute reperfusion significantly improved functional recovery and significantly increased phospho-AMPKαThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45 minutes at reperfusion recovered poorly with no phospho-AMP-activated protein kinase α activation, decreased phospho-eNOSSer633, and decreased mitochondrial cytochrome c content with increased lactate dehydrogenase release. CONCLUSIONS: Inclusion of CsA during cold storage is cardioprotective. Effects of CsA addition to the perfusate during reperfusion were time dependent, with benefits at 15 minutes but not 45 minutes of reperfusion. The toxic effect with the presence of CsA for the full 45-minute reperfusion is associated with impaired mitochondrial integrity and decreased eNOS phosphorylation.

Quaternary complexes modified from pDNA and poly-l-lysine complexes to enhance pH-buffering effect and suppress cytotoxicity.

We developed a modified complex of pDNA and poly-l-lysine (PLL) by the addition of poly-l-histidine (PLH) and γ-polyglutamic acid (γ-PGA) to enhance its pH-buffering effect and suppress cytotoxicity. The binary and ternary complexes of pDNA with PLL or/and PLH showed particle sizes of approximately 52-76 nm with cationic surface charge. The ternary complexes showed much higher gene expression than the binary complexes with PLL. The mixed solution of PLL and PLH showed higher buffering capacity than PLL solution. The high gene expression of ternary complexes was reduced by bafilomycin A1 . These results indicated the addition of PLH to PLL complexes promoted endosomal escape by enhancing the pH-buffering effect. The binary and ternary complexes showed cytotoxicity and blood agglutination because of their cationic surface charge. We therefore developed quaternary complexes by the addition of anionic γ-PGA, which was reported to decrease the toxicity of cationic complexes. In fact, quaternary complexes showed no cytotoxicity and blood agglutination. Also, quaternary complexes showed higher gene expression than ternary complexes regardless of their anionic surface charge. Quaternary complexes showed selectively high gene expression in the spleen after their intravenous administration. Thus, we successfully developed the quaternary complexes with high gene expression and no toxicity.

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 µg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives.

Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by 1H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%-50%, HPG4 showed 10-100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.

Cellular lipid metabolism is influenced by the coordination environment of copper.

Copper ions are vital to human health, and mis-trafficking of them can result in many diseases including Wilson's, Menkes', and Alzheimer's diseases. Coherent anti-Stokes Raman scattering (CARS) microscopy can be used to observe changes in lipid phenotype in a noninvasive manner and is employed here to show that copper accumulation in hepatic cells results in rapid changes in lipid storage and lipid droplet density. The increase in lipid storage is dependent on the coordination environment of the copper to which the cells are exposed and changes in toxicity, lipid phenotype, and rate of copper accumulation upon treatment vary using different Cu species.

Inherent toxicity of organ preservation solutions to cultured hepatocytes.

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 degrees C), intermediate (21 degrees C) and physiological (37 degrees C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 degrees C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 degrees C: HTK 76+/-2%, Euro-Collins 78+/-17%, HL 81+/-15%; control: Krebs-Henseleit buffer 20+/-6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.

Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators.

The amino acid histidine is an excellent buffer and is therefore included in several organ preservation solutions used in transplantation medicine. However, when used at concentrations as in these solutions, histidine has a marked injurious potential. Therefore, we here assessed the mechanism of histidine-induced cell injury and searched for ways to use the buffering power of histidine but avoid histidine toxicity. When cultured hepatocytes were incubated in HTK solution or in modified Krebs-Henseleit buffer containing 198 mM L-histidine at 37 degrees C, most cells lost viability within 3 h (LDH release 86 +/- 7% and 89 +/- 5%, respectively). This injury was accompanied by marked lipid peroxidation, and was strongly inhibited by hypoxia, by the antioxidants trolox, butylated hydroxytoluene and N-acetylcysteine and by the membrane-permeable iron chelators 2,2'-dipyridyl, 1,10-phenanthroline, LK 614, LK 616 and deferoxamine. Thus, histidine-induced cell injury appears to be mediated by an iron-dependent formation of reactive oxygen species. D-Histidine, imidazol and L-histidine methyl ester also elicited marked injury, while the N-substituted derivatives Nalpha-acetyl-L-histidine and tert-butyl-oxycarbonylhistidine and histidine-containing dipeptides showed almost no toxicity. Histidine toxicity, its iron dependence and the superiority of Nalpha-acetyl-L-histidine were also evident during/after cold (4 degrees C) incubations. Therefore, we suggest the addition of iron chelators to histidine-containing solutions, and/or replacing histidine with Nalpha-acetyl-L-histidine in organ preservation solutions.

Histidines 13 and 14 in the Abeta sequence are targets for inhibition of Alzheimer's disease Abeta ion channel and cytotoxicity.

The fact that Alzheimer's beta amyloid (Abeta) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Abeta ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Abeta channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Abeta channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Abeta cytotoxicity. These data thus contribute to the definition of the region of the Abeta sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Abeta. These data also emphasize the potential value in using inhibition of Abeta ion channel activity as an end point for Alzheimer's disease drug discovery.

Histidines 13 and 14 in the Aâ sequence are targets for inhibition of Alzheimer's disease Aâ ion channel and cytotoxicity

The fact that Alzheimer's beta amyloid (Aâ) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Aâ ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Aâ channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Aâ channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Aâ cytotoxicity. These data thus contribute to the definition of the region of the Aâ sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Aâ. These data also emphasize the potential value in using inhibition of Aâ ion channel activity as an end point for Alzheimer's disease drug discovery.

Development of a peptide reactivity assay for screening contact allergens.

Allergic contact dermatitis resulting from skin sensitization is a common occupational and environmental health problem. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurate identification of skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency; information that is pivotal in successful management of human health risks. However, even with the significant animal welfare benefits provided by the LLNA, there is still interest in the development of nonanimal test methods for skin sensitization testing. One characteristic of a chemical allergen is its ability to react with proteins prior to the induction of skin sensitization. The majority of chemical allergens is electrophilic and as such reacts with nucleophilic amino acids like cysteine or lysine. In order to determine if reactivity correlates with sensitization potential, 38 chemicals representing allergens of different potencies (weak to extreme) and nonsensitizers were evaluated for their ability to react with glutathione or three synthetic peptides containing either cysteine, lysine, or histidine. Following a 15-min reaction time for glutathione or a 24 h reaction period for the three synthetic peptides, the samples were analyzed by HPLC. UV detection was used to monitor the depletion of glutathione or the peptide following reaction. The results demonstrate that a significant correlation (Spearman correlation) exists between allergen potency and the depletion of glutathione (p = 0.001), lysine (p = 0.025), and cysteine (p = 0.020), but not histidine. The peptide with the highest sensitivity was cysteine (80.8%) whereas histidine was the least sensitive (11.5%). The data presented show that measuring peptide reactivity has utility for screening chemicals for their skin sensitization potency and thus potential for reducing our reliance on animal test methods.

Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells.

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.

pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer.

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ¿PLH¿ has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ¿PLH-g-PLL¿ was synthesized by grafting poly(L-histidine) to poly(L-lysine) ¿PLL¿. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ¿pDNA¿. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.

Long-term toxicity/carcinogenicity study of L-histidine monohydrochloride in F344 rats.

The long-term toxicity and carcinogenicity of histidine, an essential amino acid for most animal species, were examined in Fischer 344 (F344) rats. Groups of 50 males and 50 females were given L-histidine monohydrochloride (HMHC) in their diet at concentrations of 0 (control), 1.25 and 2.5% for 104 wk; these dose levels were selected on the basis of the results of a subchronic toxicity study, in which body weights were depressed and formation of sperm granulomas in the epididymis was histologically evident in males fed 5.0% HMHC. All surviving rats were killed at wk 107. Increases in red blood cell count, haemoglobin value and haematocrit level were observed in male rats given 2.5% HMHC. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in this strain of rats, and no statistically significant increase in the incidence of any tumor was found in the treated groups of either sex. Thus, it was concluded that, under the present experimental conditions, HMHC is not carcinogenic in F344 rats.

Synergistic/comutagenic action in the Ames test as an indication of irrelevant positive findings.

A number of structurally very diverse compounds which cause weak positive effects in the Ames test by evident or suspect irrelevant mechanisms is discussed. As a unifying observation we describe synergistic effects in combination with known mutagens in the responsive strains and comutagenic effects in initially unresponsive strains. We argue that the compounds enhance the formation of spontaneous (or mutagen-induced) revertant colonies by test-specific mechanisms likely to be of no relevance to multicellular eukaryotic organisms rather than possessing intrinsic genotoxic (i.e. DNa-damaging) properties in the Ames test.

Simultaneous determination of DNA double strand breaks and DNA fragment size in cultured mammalian cells exposed to hydrogen peroxide/histidine or etoposide with CHEF electrophoresis.

A CHEF (contour clamped, homogenous electric field) assay allowing the measurement of chemically-generated DNA DSBs (double strand breaks), and the simultaneous estimation of the size of the resulting double stranded DNA fragments, in a single gel run, has been developed. This method combines a very high sensitivity for detecting DNA DSBs with a very good resolution over a broad range of megabase--sized DNA. This information can be obtained in a 68 h gel run, a time which is slightly elevated as compared to the CHEF DSB assay (approximately 20 h), but dramatically reduced as compared to other CHEF protocols utilized for resolving DNA fragments of 0.2-5.7 Mb (5-14 days). Treatment with 5-10 microM etoposide or 50-100 microM hydrogen peroxide/300 microM histidine produced DNA fragments with a mean size of 7.7 x 10(5) bp (from < or = -0.2 Mb) or 4.6 x 10(6) bp (from > or = 5.7-2.2 Mb), respectively.

[13-week subchronic toxicity study of L-histidine monohydrochloride in F344 rats].

A 13-week subchronic toxicity study of L-histidine monohydrochloride was performed in male and female F344 rats at dose levels of 0, 0.31, 0.62, 1.25, 2.5 and 5.0% in the diet, to determine the maximum tolerable dose (MTD) for subsequent investigation of carcinogenicity. Rats were randomly allocated to 6 groups, each consisting of 10 males and 10 females. No animals died during the administration period. Suppression of body weight gain and decrease in food consumption were observed in males of the 5.0% group along with hematological examination as revealed by increases in hemoglobin volume and hematocrit. Serum biochemical examination demonstrated increased levels of BUN and creatinine in females of the 5.0% group, and increased level of BUN in females of the 1.25% group. Histopathologically, sperm granulomas in the epididymis were found in half of the 5.0% group males. Based on the results of the present study, it was concluded that the MTD of L-histidine monohydrochloride is 2.5% in diet, because the dietary dose level of 5.0% proved to exert significant toxicity reflected in suppression of body weight gain and formation of sperm granulomas.

Enhancement by L-histidine of nickel(II)-induced DNA-protein cross-linking and oxidative DNA base damage in the rat kidney.

Formation of DNA-protein cross-links and oxidatively damaged DNA bases was investigated with the use of alkaline elution and gas chromatography/mass spectrometry techniques in the nuclei from kidneys of rats 3 and 18 h after a single iv injection of the NiII(His)2 complex (NiHis), nickel(II) acetate (NiAcet), or L-histidine (His). Administration of 20 mumol of NiHis/kg body wt caused the formation of DNA-protein cross-links and significantly increased levels of oxidatively damaged DNA bases, including 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua; 3.5-fold vs the control value) 3 h postinjection and 8-oxoguanine (2.6-fold), cytosine glycol (2.5-fold), 8-oxoadenine (2-fold), and FapyGua (1.9-fold) 18 h postinjection. Injection of 20 mumol of NiAcet/kg body wt enhanced the cross-linking to a lesser extent than NiHis and did not significantly increase the amounts of modified DNA bases over the control levels. Forty micromoles of His per kilogram body wt alone caused a marked DNA-protein cross-linking effect and increased the amount of 4,6-diamino-5-formamidopyrimidine (2-fold vs the control) 3 h, but not 18 h, after treatment. The DNA base derivatives found were typical products of hydroxyl radical (.OH) attack on DNA. Formation of the cross-links may also be attributed to .OH, although other mechanisms, e.g., formation of ternary complexes of Ni(II), cannot be excluded. The present in vivo study confirms the conclusion of our former in vitro experiments that His enhances Ni(II)-mediated oxidative damage to DNA and chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)