A novel DTPA cross-linking of hyaluronic acid and metal complexation thereof

Macromolecular conjugates of a natural polysaccharide, hyaluronic acid, with diethylenetriaminepentaacetic acid (DTPA)—metal complexes were synthesized and characterized by FTIR, NMR, SEC-MALLS and ICP analysis. Several parameters of the cross-linking reaction as molecular weight of starting HA, temperature, equivalent of DTPA bis-anhydride, concentration of HA, presence of transacylation catalyst DMAP and reaction time were studied. The mechanism for the reaction was suggested and relationship between the molecular weight assigned by SEC-MALLS, reaction parameters and rheological properties of the final cross-linked products were investigated.     

Characterization of the chemical degradation of hyaluronic acid during chemical gelation in the presence of different cross-linker agents

Dynamic light scattering (DLS) and rheological experiments have been performed on semidilute aqueous hyaluronic acid (HA) solutions during the chemical cross-linking process with a water-soluble carbodiimide (WSC) to produce a hydrogel. The formation and destruction of the gel are characterized. The results suggest that the gel is cross-linked via ester linkages and at later stage in the process, the omnipresent hydrolysis of interpolymer ester linkages and glycosidic bonds prevails, leading to disruption of the gel. The process of forming and breaking the gel is affected by the cross-linker concentration and pH. The cross-linking of HA with WSC in the presence of L-lysine methyl ester produced a gel with a longer time of gelation and the degradation of the gel was prolonged because of the more stable amide bond formation as the cross-link. By using the Ugi multicomponent condensation reaction, interpolymer cross-linking occurs via the formation of amide linkages and a stable gel evolves, which is only slightly degraded over an extended time window. DLS measurements on HA solutions with WSC show the emergence of a long-time power-law tail in the correlation functions at conditions both before and beyond the viscosity maximum. At a late stage in the gel-breakage regime, the power-law profile of the decay disappears and the long-time tail of the correlation function can be portrayed by a stretched exponential. The findings indicate that the power-law feature is associated with the confinement of chain dynamics and anomalous diffusion in the system. At later times, the connectivity is lost due to fragmentation of the network, and the long-time stretched exponential decay in the correlation function reflects the relaxation of clusters of various sizes.     

Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats

Female Wistar rats (100 weeks old) were divided into two groups; one group was fed a high-cholesterol diet (HC) and the other a high-cholesterol diet plus docosahexaenoic acid (HC-fed DHA rats). Fatty acid concentrations in brain tissues were analyzed by gas chromatography. In the HC-fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx) increased in the cerebrum but not in the brainstem or cerebellum. The rate of increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with that in the HC animals (p < 0.05). In the cerebrum of the HC-fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.     

Influence of soil waterlogging on subsequent plant growth and trace metal content

Summary The uptake of trace metals by two plant species (French bean and maize) has been measured on two soils subjected to various waterlogging regimes. Uptake of both manganese and iron was increased due to soil waterlogging, although reoxidation of the soil affected iron more than manganese. Zinc and copper uptake was influenced by a species factor; French bean (Phaseolus vulgaris) showed preferential uptake of zinc, whereas maize (Zea mays) took up copper preferentially. Uptake of cobalt by both species was increased due to waterlogging, following the pattern of manganese.The abilities of these species to take up trace metals from soil followed the pattern predicted by selective extraction of soil for manganese, iron and cobalt, but not for zinc and copper.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

Medium-chain fatty acids accumulating in MCAD deficiency elicit lipid and protein oxidative damage and decrease non-enzymatic antioxidant defenses in rat brain

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent disorder of fatty acid oxidation with a similar prevalence to that of phenylketonuria. Affected patients present tissue accumulation of the medium-chain fatty acids octanoate (OA), decanoate (DA) and cis-4-decenoate. Clinical presentation is characterized by neurological symptoms, such as convulsions and lethargy that may develop into coma and sudden death. The aim of the present work was to investigate the in vitro effect of OA and DA, the metabolites that predominantly accumulate in MCADD, on oxidative stress parameters in rat cerebral cortex homogenates. It was first verified that both DA and OA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation) and decreased the non-enzymatic antioxidant defenses, measured by the decreased total antioxidant capacity. DA also enhanced carbonyl content and oxidation of sulfhydryl groups (protein damage) and decreased reduced glutathione (GSH) levels. We also verified that DA-induced GSH decrease and sulfhydryl oxidation were not observed when cytosolic preparations (membrane-free supernatants) were used, suggesting a mitochondrial mechanism for these actions. Our present data show that the medium-chain fatty acids DA and OA that most accumulate in MCADD cause oxidative stress in rat brain. It is therefore presumed that this pathomechanism may be involved in the pathophysiology of the neurologic symptoms manifested by patients affected by MCADD.     

Performance of the main downstream operations on hyaluronic acid purification

Hyaluronic acid (HA), or hyaluronan, is a natural polyelectrolyte, ubiquitous in human tissues. Exogenous HA has been a valuable material due to its wide range of medical applications, such as in osteoarthritis treatment, ophthalmic surgery, adhesion prevention after surgeries and wound healing, as well as cosmetic applications. However, to ensure the physicochemical and biological properties, a purity near to 99 % is a primary requirement, aiming clinical applications. To achieve this goal, various downstream operations have been used, aiming HA concentration, separation and purification. Precipitation with organic solvents has been a common operation in most purification processes, combined with other downstream operations such as precipitation with quaternary salts, filtration, adsorption and ion exchange. This work presents an updated review of HA purification, emphasizing the performance of the main downstream operations used to achieve highly purified HA, in the period from 1970 to 2019. We conclude that, in the majority of the published works, there is a lack of studies regarding the operational conditions, as well as an absence of the purification percentage development during the processes.     

Neuroprotective effects of the antioxidant action of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against ischemic neuronal damage in the brain

Ischemia is characterized by oxidative stress and changes in the antioxidant defense system. Our recent in vitro study showed that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects cortical astrocytes against oxidative stress. In the current study, we examined the effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride on ischemia-induced neuronal damage in a gerbil ischemia/reperfusion models. Extensive neuronal death in the hippocampal CA1 area was observed 4 days after ischemia/reperfusion. Intraperitoneal injection of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (0.3 mg/kg body weight) significantly prevented neuronal death in the CA1 region of the hippocampus in response to transient forebrain ischemia. 2-Cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride administration reduced ischemia-induced increases in reactive oxygen species levels and malondialdehyde content. It also attenuated the associated reductions in glutathione level and superoxide dismutase, catalase, and glutathione peroxidase activities. Taken together, our results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against ischemia-induced neuronal damage by reducing oxidative stress through its antioxidant actions. [BMB Reports 2013; 46(7):370-375]     

Attenuation of fluoride-induced hepatorenal oxidative stress by ferulic acid in vivo: An approach with in-silico analysis and interaction informatics of ferulic acid

BackgroundChronic fluoride toxicity induces oxidative strain and lipid peroxidation and imparts deleterious effects on human metabolic organs.AimThe present study aimed to expose the defensive impact of ferulic acid against sodium fluoride (NaF) induced hepatorenal dysfunction at the biochemical and antioxidative systems.MethodsIn-vivo. Rats were arbitrarily separated into five groups as control, sodium fluoride-treated (200 ppm kg ⁻¹), vitamin C -as a positive control, and FA co-administered groups with 10 mg kg ⁻¹ and 20 mg kg ⁻¹ body weight for 56 days. In the present investigation, we measured antioxidant enzymes, superoxide dismutase, catalase, and lactate dehydrogenase by electrozymographic and spectrophotometric methods. Biochemical assessment of TBARS, conjugated diene, and different serum biomarkers was done for liver and kidney functionality tests.In-silico. An in-silico study was conducted through a molecular docking experiment to evaluate the binding potentiality of FA by employing AutoDock Vina [version 1.5.6] to overcome the abnormality in the activities of catalase, and superoxide dismutase in NaF promoted toxicity of hepatorenal system.In-vitro. An in vitro biochemical experiment was conducted to support the in-silico study.ResultsSuperoxide dismutase and catalase were decreased in the intoxicated rat. Ferulic acid (FA) as an antioxidant remarkably defended the NaF-mediated deterioration of the antioxidative status in the hepatorenal system, lowering lipid peroxidation products, malondialdehyde, and conjugated diene. Serum biomarkers, ALT, AST, ALP, urea, and creatinine increased in the intoxicated group than in control. Ferulic acid significantly neutralized the ill effects of NaF on serum lipid profile. In-silico analysis hypothesized the strong interaction of FA with the active side of catalase and superoxide dismutase that prevented the binding of NaF at the active site of these mentioned enzymes and this was further validated by in-vitro assay.ConclusionHowever, FA modulates free radical generation and protected these metabolic organs against sodium fluoride-induced injury.     

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive species (ROS), at concentrations as low as 50–100 μM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porhyria patients. Removal of Ca²⁺ (10 μM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swellling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca²⁺. Addition of MgCl₂ at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca²⁺ efflux. This indicates that Mg²⁺ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.     

Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.     

Effect of indole acetic acid administration on the neutrophil functions and oxidative stress from neutrophil, mesenteric lymph node and liver

This study was done to investigate the effect of the in vivo administration of indole acetic acid (IAA) on the neutrophil function, the activities of antioxidants enzymes in neutrophils, the mesenteric lymph node and on the oxidative stress in liver and plasma. The animals received subcutaneous administration of IAA in a phosphate-buffered saline (the control group received only the phosphate-buffered saline). The other groups received IAA at concentrations of 1 mg (T1), 2 mg (T2) and 18 mg (T3) per kg of body mass per day. Administration of IAA in both treatments T2 and T3 promoted a significant rise in the phagocytic capacity of neutrophils (by 51%), in comparison with the control. Another alteration was observed in antioxidant enzyme activities of the neutrophil and lymph node. But in the liver, the treatments imposed a significant decrease in the activity of catalase of 19% and 30% for T2 and T3, respectively, in comparison with the control. A similar effect was observed in the activity of hepatic glutathione peroxidase for T3 where a significant decrease of 31%, compared with the control, was obtained. The IAA did not show another significant alteration of the activities of superoxide dismutase and glutathione reductase activities in liver. The hepatic lipid peroxidation level, available by reactive products with thiobarbituric acid, has shown a significant decrease of 27% and 29% with T1 and T3 respectively, in comparison with the control. IAA treatment did not show a significant alteration in reduced glutathione contents in comparison with the liver and plasma controls. In conclusion, the IAA administration has a good potential animal utilization for increasing the phagocytic capacity with no prooxidant effect.     

The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis

Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.     

Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn²⁺ or Co²⁺ ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn²⁺ ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn²⁺ improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn²⁺ ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co²⁺ ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern–Vollmer constants of 10–300 μM.     

Some effects of metal ions on DNA structure and genetic information transfer

The reaction of metal ions with nucleic acids can lead to a variety of dramatic effects on nucleic acid structure, e.g., crosslinking of the polymer strands, degradation to oligomers and monomers, stabilization or destabilization, and the mispairing of bases. These effects have important implications for genetic information transfer. Metal ions are involved in many aspects of this transfer; we are presently concerned with the effect of metal ions on the orientation of the active site of RNA polymerase. Many of the effects of metal ions on nucleic acid structure involve changes in the conformation of the macromolecules. We have found that conditions that have been used to convert B DNA to Z DNA lead to at least two other conformational changes, and phase diagrams delineate the realms of stability of each of the forms. We have carried out a number of studies that demonstrate that the conversion of B to Z DNA is very closely correlated with a substantial decrease in the ability of the DNA to act as a template for RNA synthesis. A portion of this paper has been taken from another paper on “Changes of Biological Significance Induced by Metal Ions in the Structure of Nucleic Acids,” published in Annali dell' lstituto Superiore di Sanita.     

Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum

This paper aims to determine the changes in reactive oxygen species (ROS) and the responses of the lily (Lilium longiflorum L.) antioxidant system to short-term high temperatures. Plants were exposed to three levels of heat stress (37°C, 42°C, 47°C) for 10 h when hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rate along with membrane injury indexes, and changes in antioxidants were measured. Compared with the control (20°C), electrolyte leakage and MDA concentration varied slightly after 10 h at 37°C and 42°C, while increased significantly at 47°C. During 10 h at 37°C and 42°C, antioxidant enzyme activities, such as SOD, POD, CAT, APX and GR, were stimulated and antioxidants (AsA and GSH concentrations) maintained high levels, which resulted in low levels of O₂⁻ and H₂O₂ concentration. However, after 10 h at 47°C, SOD, APX, GR activities and GSH concentration were similar to the controls, while POD, CAT activities and AsA concentration decreased significantly as compared with the control, concomitant with significant increase in O₂⁻ and H₂O₂ concentrations. In addition, such heat-induced effects on antioxidant enzymes were also confirmed by SOD and POD isoform, as Cu/ZnSOD maintained high stability under heat stress and the intensity of POD isoforms reduced with the duration of heat stress, especially at 47°C. It is concluded that in lily plants, the oxidative damage induced by heat stress was related to the changes in antioxidant enzyme activities and antioxidants.     

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.     

Formation of phosphatidic acid in stressed mitochondria

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu²⁺(Fe²⁺)/H₂O₂/ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu²⁺/H₂O₂/and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO-radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.     

Effects of metal ions on myrosinase activity and the formation of sulforaphane in broccoli seed

Effects of six metal ions on the formation of sulforaphane and the liberation of glucose upon hydrolysis of glucoraphanin by myrosinase at neutral pH were studied. The yields of sulforaphane and glucose were determined by HPLC. Copper ion and magnesium ion decreased the yields of sulforaphane and glucose. Ferrous ion and ferric ion inhibited the formation of sulforaphane, but had no effects on the liberation of glucose. Calcium ion increased the yield of glucose liberation, but inhibited the formation of sulforaphane. Only zinc ion was beneficial to the liberation of glucose and accelerated the formation of sulforaphane at initial reaction intervals.     

Sporicidal action of peracetic acid and protective effects of transition metal ions

Although peracetic acid (PAA) is used widely for cold sterilization and disinfection, its mechanisms of sporicidal action are poorly understood. PAA at high concentrations (5–10%) can cause major loss of optical absorbance and microscopically-visible damage to bacterial spores. Spores killed by lower levels of PAA (0.02–0.05%) showed no visible damage and remained refractile. Treatment of spores ofBacillus megaterium ATCC 19213 with PAA at concentrations close to the lethal level sensitized the cells to subsequent heat killing. In addition, PAA was found to act in concert with hypochlorite and iodine to kill spores. Antioxidant sulfhydryl compounds or ascorbate protected spores against PAA killing. Trolox, a water-soluble form of -tocopherol, was somewhat protective, while other antioxidants, including -tocopherol, urate, bilirubin, ampicillin and ethanol were not protective. Chelators, including dipicolinate, were not protective, but transition metal ions, especially the reduced forms (Co²⁺, Cu⁺ and Fe²⁺) were highly protective. The net conclusions are that organic radicals formed from PAA are sporicidal and that they may act as reducing agents for spores that are normally in a highly oxidized state, in addition to their well known actions as oxidizing agents in causing damage to vegetative cells.