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Preparation for human study of pesticide applicators: sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to selected fumigants.

In preparation for a human study of worker exposure to grain fumigants and pesticides, we decided to screen commonly used fumigants for genotoxic effects in vitro. This research strategy was employed to test the possibility that structurally simple chemicals might have similar genotoxic properties in vivo and in vitro. As a first step, we designed our in vitro protocol to mimic to the extent possible, a single in vivo exposure of lymphocytes to fumigants. Go lymphocytes were treated with different doses of carbon tetrachloride, carbon disulfide, methyl bromide, chloropicrin, and melathion with and without addition of rat liver homogenate for 1/2 hour, washed free of toxicant, and stimulated with PHA. After culture, the prepared slides were studied for chromosome aberrations and SCEs. Malathion, methyl bromide, and chloropicrin significantly induced SCEs without S-9. Carbon disulfide alone required S-9 for significant SCE induction. Chromosome aberrations were significantly increased by malathion and methyl bromide. Carbon tetrachloride failed to induce SCEs or chromosome aberrations with or without S-9. We concluded from these preliminary studies and other comparable work that the fumigants studied here may be less likely to express genotoxicity in terms of SCEs or chromosome aberrations than ethylene oxide or phosphine given a single short-term in vivo exposure. The final design of our human study was altered to focus on seasonal worker exposure rather than on a single exposure event.

Adult↗

Free-air fumigation of mature trees. A novel system for controlled ozone enrichment in grown-up beech and spruce canopies.

A novel system for continuous and controlled free-air fumigation of mature tree canopies with ozone is described. Ozone generated from oxygen is diluted with air in a pressurized tank and conducted into the canopies by a system of 100 PTFE tubes hanging down from a grid fixed above the crowns. With 45 calibrated outlets per tube providing a constant flow of 0.3 l/min each, a total volume of about 10*10*15 m3 comprising 5 beech and 5 spruce canopies is fumigated. The spatial ozone distribution in the fumigated volume as well as surrounding reference tree canopies is controlled by continuous measuring instruments installed at 4 levels and a dense array of passive samplers. The system will later be used for CO2 fumigation as well. Results of the first year of continuous operation, with 2 * ambient ozone levels having been achieved, are reported.

Forestry↗

Association of fallout radiocesium with soil constituents: effect of sterilization of forest soils by fumigation with chloroform.

The effect of soil sterilization by chloroform fumigation on the release of fallout radiocesium incorporated in the fungal biomass of the organic layer of two forest soils was investigated by applying a sequential extraction procedure for radiocesium. The amount of the biomass in all soil samples was estimated by determination of the dissolved organic carbon (DOC) before and after fumigation, and qualitatively also by the ergosterol test. The five fractions obtained by sequential extraction (modified Tessier procedure) were: (I) easily exchangeable, (II) bound to oxides, (III) bound to organic matter, (IV) persistently bound, (V) residual. For the samples from the soil under spruce trees, no significant effects were apparent in any of these five fractions as a result of chloroform fumigation, indicating that the amount of radiocesium in the biomass of this soil was obviously negligibly small compared with the radiocesium associated with other soil constituents. The results obtained for the soil samples from the beech stand, however, reveal that the destruction of the biomass by chloroform fumigation modified considerably the extent of the association (i.e., binding) of radiocesium with the various other soil constituents (especially the clay minerals). As a result of this rapid redistribution of radiocesium released by the fungal biomass, it is not possible, in general, to attribute the observed increase of radiocesium in fraction I (easily exchangeable) after soil sterilization quantitatively to radiocesium released by the biomass. A reliable method to determine the amount of radiocesium incorporated in the fungal biomass of the soil samples which also contain clay minerals has, therefore, still to be developed.

Biomass↗

Nutritional and toxicological effects of long-term ingestion of phosphine-fumigated diet by the rat.

The fumigation of stored foodstuffs with phosphine (PH3) is likely to become widely used in the future because of its technological efficiency and the rapid desorption of the fumigant. In a long-term feeding study of a phosphine-fumigated diet, rats were monitored for weight gain, food intake, plasma chemistry, haematology and urinary changes. Histopathological studies, including organ-weight determinations, were carried out after treatment of the rats for 1 and 2 yr. The results show that ingestion of a phosphine-fumigated diet by the rat for 2 yr does not cause any marked modification of growth, food intake, nitrogen balance, body composition, functional behaviour or the incidence or type of tumours.

Animals↗

Review of the toxicology of carbonyl sulfide, a new grain fumigant.

Carbonyl sulfide (COS) is a new grain fumigant which has been developed to replace methyl bromide, being phased out due to its ozone depletion properties, and to supplement phosphine gas which is experiencing increased insect resistance. Treatment of commodities with COS, a highly effective fumigant, results in residues that are near or indistinguishable to natural background levels of this compound. COS is a naturally occurring gas, being the predominant sulfur moiety in the atmosphere, occurs naturally in food and is a normal by-product of mammalian aerobic metabolism. COS has low acute inhalational toxicity but with a steep dose response curve; COS is neither genotoxic nor a developmental toxicant but does reversibly impair male fertility. Prolonged, repeated exposure to COS is likely to present similar neurotoxicity hazards to that of the structurally and toxicologically related compound carbon disulfide. Although the occupational risks presented by COS as a fumigant of bulk grain are significant, these are, as they have been for a considerable time for phosphine and methyl bromide, manageable by good occupational safety practices. Consideration may need to be given to scrubbing of ventilated COS and its breakdown product hydrogen sulfide, at the completion of fumigation to minimise worker and bystander exposure. In terms of classical regulatory toxicology studies, the available database for COS is deficient in many aspects and registration in most jurisdictions will depend on sound scientific argument built upon the totality of the existing scientific data as there are strong arguments supporting the registration of this compound.

Animals↗

Insecticidal and fumigant activities of Cinnamomum cassia bark-derived materials against Mechoris ursulus (Coleoptera: attelabidae).

The insecticidal and fumigant activities of Cinnamomum cassia (Blume) bark-derived materials against the oak nut weevil (Mechoris ursulus Roelofs) were examined using filter paper diffusion and fumigation methods and compared to those of the commercially available Cinnamomum bark-derived compounds (eugenol, salicylaldehyde, trans-cinnamic acid, and cinnamyl alcohol). The biologically active constituent of the Cinnamomum bark was characterized as trans-cinnamaldehyde by spectroscopic analysis. In a test with the filter paper diffusion method, trans-cinnamaldehyde showed 100 and 83.3% mortality at rates of 2.5 and 1.0 mg/filter paper, respectively. At 2.5 mg/paper, strong insecticidal activity was produced from eugenol (90.0% mortality) and salicylaldehyde (88. 9%), whereas trans-cinnamic acid revealed moderate activity (73.3%). At 5 mg/paper, weak insecticidal activity (50.0%) was produced from cinnamyl alcohol. In a fumigation test, the Cinnamomum bark-derived compounds were much more effective against M. ursulus larvae in closed cups than in open ones. These results indicate that the insecticidal activity of test compounds was attributable to fumigant action, although there is also significant contact toxicity. As a naturally occurring insect-control agent, the Cinnamomum bark-derived materials described could be useful as a new preventive agent against damage caused by M. ursulus.

Aldehydes↗

Respiratory exposure of grain inspection workers to carbon tetrachloride fumigant.

Carbon tetrachloride (often mixed with carbon disulfide or ethylene dichloride) is a common constituent of liquid grain fumigants. Applied as liquids, these mixtures volatilize and achieve vapor concentrations sufficient to control insect infestations in stored grains. Absorbed grain desorbs fumigant components after the fumigation period, and it then becomes a source of exposure to workers who handle fumigated grain. Carbon tetrachloride meets the EPA's risk criteria for hepatotoxicity, nephrotoxicity and oncogenicity, and it has been under regulatory review since 15 October 1980. Present OSHA standards for CCl4 are a time-weighted average (TWA) of 10 ppm and an acceptable ceiling of 25 ppm. ACGIH and NIOSH have recommended lowering the OSHA standard. The point at which peak exposure occurs during the grain inspection process has been identified as the off odor test in which the inspector smells the grain sample for rancidity, sourness, etc. Ambient concentrations of CCl4 in 7750 grain samples submitted for inspection were determined by colorimetric tube, and these concentrations were an estimate of peak grain inspector exposure to CCl4. The average ambient concentration of CCl4 per grain sample was 1.69 ppm +/- 8.35. Approximately 380 TWA CCl4 exposures for grain inspection workers were determined by using passive dosimeters. All TWA exposures were less than 2 ppm. Variables are examined, such as location of work, grain type, time of year and grain transportation vehicle--all of which are known to the sampler or inspector before performing their job functions and which affect potential exposure.

Agricultural Workers' Diseases↗

Indoor winter fumigation of Apis mellifera (Hymenoptera: Apidae) colonies infested with Varroa destructor (Acari: Varroidae) with formic acid is a potential control alternative in northern climates.

Formic acid treatment for the control of the ectoparasitic varroa mite, Varroa destructor Anderson & Trueman, infesting honey bee, Apis mellifera L., colonies is usually carried out as an in-hive outdoor treatment. This study examined the use of formic acid on wintered colonies kept indoors at 5 degrees C from 24 November 1999 to 24 March 2000. Colonies were placed in small treatment rooms that were not treated (control) or fumigated at three different concentrations of formic acid: low (mean 11.9 +/- 1.2 ppm), medium (mean 25.8 +/- 1.4 ppm), or high (mean 41.2 +/- 3.3 ppm), for 48 h on 22-24 January 2000. Queen bee, worker bee, and varroa mite mortality were monitored throughout the winter, and tracheal mite, Acarapis woodi (Rennie), prevalence and mean abundance of nosema, Nosema apis Zander, spores were assessed. This study revealed that formic acid fumigation of indoor-wintered honey bees is feasible and effective. The highest concentration significantly reduced the mean abundance of varroa mites and nosema spores without increasing bee mortality. Tracheal mite prevalence did not change significantly at any concentration, although we did not measure mortality directly. The highest concentration treatment killed 33.3% of queens compared with 4.8% loss in the control. Repeated fumigation periods at high concentrations or extended fumigation at low concentrations may increase the efficacy of this treatment method and should be tested in future studies. An understanding of the cause of queen loss and methods to prevent it must be developed for this method to be generally accepted.

Acari↗

Personal air sampling and biological monitoring of occupational exposure to the soil fumigant cis-1,3-dichloropropene.

OBJECTIVES: To assess exposure of commercial application workers to the nematocide cis-1,3-dichloropropene (cis-DCP). METHODS: The study was conducted during the annual application season, August to 15 November, in the starch potato growing region in The Netherlands. 14 Application workers collected end of shift urine samples on each fumigation day (n=119). The mercapturic acid metabolite N-acetyl-S-(cis-3-chloro-2-propenyl)-L-cysteine (cis-DCP-MA) in urine was used for biological monitoring of the cis-DCP uptake. Inhalatory exposure was assessed by personal air sampling during a representative sample (n=37) of the fumigation days. Extensive information was collected on factors of possible relevance to the exposure and the application workers were observed for compliance with the statutory directions for use. The inhalatory exposure during all fumigation days was estimated from the relation between the personal air sampling data and the biological monitoring data. Exposure levels were correlated with the general work practice. The fumigation equipment and procedures were in accordance with the statutory directions of use, with the exception of the antidrip systems. Two antidrip systems were used: antidrip nozzles or a compressed air system. RESULTS: The geometric mean exposure of the application workers was 2.7 mg/m(3) (8 hour time weighted average); range 0.1-9.5 mg/m(3). On 25 days (21%) the exposure exceeded the Dutch occupational exposure limit (OEL) of 5 mg/m(3). This could mainly be explained by prolonged working days of more than 8 hours. The general work practice of the application workers was rated by the observers as good or poor. No difference in exposure to cis-DCP was found in the use of none, one, or two antidrip systems. Malfunctioning of the antidrip systems and lack of experience with the compressed air system were identified as possible causes for the lack of effectiveness of these antidrip systems. The use of personal protection was not always in accordance with the statutory directions of use. Dermal exposure to liquid cis-DCP was found four times during repair and maintenance, but the biological monitoring data did not suggest a significant increase in cis-DCP uptake. CONCLUSIONS: The application of cis-DCP in the potato growing industry can be performed at exposure concentrations below the Dutch OEL of 5 mg/m(3) if the working days are limited to 8 hours. An injector equipped with either kind of antidrip system which is in good working order, as well as the consistent use of personal protection in accordance with the statutory directions of use, may ensure exposure concentrations below the Dutch OEL.

Adult↗

Container fumigation as a quarantine treatment for Anoplophora glabripennis (Coleoptera: Cerambycidae) in regulated wood packing material.

Anoplophora glabripennis Motschulsky, an Asian cerambicid beetle, was first found in the United States in 1996 and was likely introduced into the United States through infested wood packing materials and dunnage. Methyl bromide (MeBr) fumigation is an internationally accepted treatment for such wood-boring beetles, but the use of MeBr is scheduled for reduction or elimination as alternatives become available. The use of MeBr can be reduced by more efficient and technically sound fumigation techniques, including good circulation and proper loading. Concentration x time products (CxT) reported for A. glabripennis in solid wood timbers were used to test estimated doses in container fumigations conducted in Tianjin and Shanghai, China, during years 2002-2003. We found that CxT products observed were generally adequate and would support reduced doses of MeBr at temperatures intermediate (10.0 and 15.6 degrees C) to the Animal and Plant Health Inspection Service schedule of 80 g and 48 g/m3 at > or =4.4 and > or = 21.1degrees C, respectively. The use of fans is recommended, and loading of the containers should never reach 100% to allow for better circulation and desorption. Proper use of fans will allow for better aeration and increase safety to inspectors and consignees of containerized fumigated commodities.

Animals↗

Health effects associated with sulfuryl fluoride and methyl bromide exposure among structural fumigation workers.

OBJECTIVES: This study assessed the health effects associated with occupational exposure to methyl bromide and sulfuryl fluoride among structural fumigation workers. METHODS: A cross-sectional study of 123 structural fumigation workers and 120 referents in south Florida was conducted. Nerve conduction, vibration, neurobehavioral, visual, olfactory, and renal function testing was included. RESULTS: The median lifetime duration of methyl bromide and sulfuryl fluoride exposure among workers was 1.20 years and 2.85 years, respectively. Sulfuryl fluoride exposure over the year preceding examination was associated with significantly reduced performance on the Pattern Memory Test and on olfactory testing. In addition, fumigation workers had significantly reduced performance on the Santa Ana Dexterity Test of the dominant hand and a nonsignificantly higher prevalence of carpal tunnel syndrome than did the referents. CONCLUSIONS: Occupational sulfuryl fluoride exposures may be associated with subclinical effects on the central nervous system, including effects on olfactory and some cognitive functions. However, no widespread pattern of cognitive deficits was observed. The peripheral nerve effects were likely caused by ergonomic stresses experienced by the fumigation workers.

Adult↗

Using surface water application to reduce 1,3-dichloropropene emission from soil fumigation.

High emissions from soil fumigants increase the risk of detrimental impact on workers, bystanders, and the environment, and jeopardize future availability of fumigants. Efficient and cost-effective approaches to minimize emissions are needed. This study evaluated the potential of surface water application (or water seal) to reduce 1,3-dichloropropene (1,3-D) emissions from soil (Hanford sandy loam) columns. Treatments included dry soil (control), initial water application (8 mm of water just before fumigant application), initial plus a second water application (2.6 mm) at 12 h, initial plus two water applications (2.6 mm each time) at 12 and 24 h, standard high density polyethylene (HDPE) tarp, initial water application plus HDPE tarp, and virtually impermeable film (VIF) tarp. Emissions from the soil surface and distribution of 1,3-D in the soil-gas phase were monitored for 2 wk. Each water application abruptly reduced 1,3-D emission flux, which rebounded over a few hours. Peak emission rates were substantially reduced, but total emission reduction was small. Total fumigant emission was 51% of applied for the control, 46% for initial water application only, and 41% for the three intermittent water applications with the remaining water treatment intermediate. The HDPE tarp alone resulted in 45% emission, while initial water application plus HDPE tarp resulted in 38% emission. The most effective soil surface treatment was VIF tarp (10% emission). Surface water application can be as effective, and less expensive than, standard HDPE tarp. Frequent water application is required to substantially reduce emissions.

Agriculture↗

Effects of formaldehyde fumigation of housing on carotenoid pigmentation in three breeds of chickens.

Three breeds of chickens (two broiler breeds, A and B, and one layer breed, C) were raised from 1 day to 3 wk of age in either standard housing or housing additionally fumigated with formaldehyde. Groups of chicks from each breed in both housings received either regular poultry starter ration or this ration supplemented with marigold meal. At 3 wk of age, chicks of all three breeds had higher (111, 113, and 115%) mean body weights when raised in fumigated housing. There was no significant effect of supplemental carotenoids on body weight. Chicks of all three breeds raised in fumigated housing had significantly higher plasma carotenoid concentrations when fed either regular or supplemented feed, and, with the exception of Breed A on regular feed, also had significantly increased skin carotenoid values. No significant differences among breeds in plasma or skin carotenoid values were seen in chicks fed regular ration. However, significant differences were seen among groups of chicks fed supplemental carotenoids. The layer breed, C, had the highest percentage increase in carotenoid pigmentation when fed the carotenoid-supplemented diet in fumigated housing.

Animal Feed↗

Environmental fate of methyl bromide as a soil fumigant.

The great variation among results of recent experiments measuring the total emission of MeBr from fields implies that many factors influence MeBr transport and transformation in the soil-water-air system and its ultimate loss from the soil surface. It has been demonstrated that variables related to application methods (e.g., injection depth, use and type of surface tarp), soil properties (e.g., water content, bulk density, soil organic matter), and climatic conditions (e.g.. air temperature, wind speed, barometric pressure) have pronounced effects on MeBr volatilization following soil injection. The following conclusions can be drawn from this experimental information. Tarping consistently, increased the residence time and concentration of MeBr residing in the soil. Prolonged retention of MeBr in the soil resulted in more extensive degradation and reduced cumulative emissions. Research indicates that the polyethylene film typically used for the surface cover is relatively permeable to MeBr and allows significant emissions compared to virtually impermeable plastic films. This effect is more pronounced during periods of high temperature. Soil type, soil water content, and bulk density are important factors affecting MeBr transport and transformation in soil, which ultimately affect volatilization. The total volatilization from a soil with high organic matter content may be drastically reduced relative to that from a low organic matter soil. Amendment of the surface soil with organic matter or nucleophilic compounds that promote increased degradation may offer another method for reducing volatilization. MeBr volatilization may also be decreased by increasing soil water content and bulk density, mainly because of the reduced gas-phase diffusion resulting from reduced soil air-filled porosity. To minimize volatilization, MeBr should be applied during periods of cool temperature, injected relatively deep in organic-rich, moist soil, and the soil surface packed and tarped immediately after the application. Depending on site-specific conditions, a new high-barrier plastic should be used. Injecting MeBr during periods of warm temperature, at a shallow depth in dry, loose soil without the use of low-permeability plastic barriers, will likely result in maximum volatilization rates and therefore should be discouraged. Before adopting any new emission reduction technology, the pest control characteristics of the new methodology should be assessed under soil and environmental conditions typical of the region to optimize efficacy while minimizing environmental contamination. There is considerable current scientific evidence indicating that eliminating MeBr use for soil fumigation may not have a significant impact on stratospheric ozone depletion. Management practices can and have been developed that essentially eliminate atmospheric emissions of MeBr and other fumigant compounds following soil application. Some scientists have suggested that there are natural buffers and various unknown sources of MeBr that make it impossible to ascertain that eliminating soil fumigation with MeBr will significantly improve stratospheric ozone levels. It is quite certain, however, that the phase-out will make it much more difficult for growers to economically provide an adequate and healthful food supply in the U.S. and elsewhere in the world. As the phase-out date approaches, there remains a great need for information about MeBr and stratospheric ozone depletion. Stratospheric ozone must be protected, but recent experiments suggest that it can be protected while still allowing MeBr to be used for soil fumigation. A new approach may be warranted in which state and federal regulations recognize that every chemical is a potential environmental contaminant, depending on the properties of the chemical and the environmental conditions prevailing following its application. Ideally, regulations should incorporate incentives to develop technology that minimizes the likelihood that a chemical becomes an environmental and/or public health problem. Rather than instituting an irrevocable ban, allowing for a suspension of chemical use until the appropriate technology is developed to control the undesirable characteristic(s) of the chemical use would provide much more flexibility to growers and may enhance environmental protection by adopting a proactive approach in which growers, chemical manufacturers, regulators, and the public can have confidence.

Adsorption↗

Effects of long-term ozone fumigations on growth and gas exchange of Fraser fir seedlings.

Fraser fir seedlings from two seed sources in the Southern Appalachians (Mt Mitchell, North Carolina, a declining population; and Mt Rogers, Virginia, a relatively healthy population) were subjected to long-term (2.5 years) intermittent ozone fumigations (0.025, 0.070, and 0.150 ppm) while being grown through five growth cycles in an accelerated-growing regime. Fumigations took place during bud break, stem elongation and bud set. Following each growing cycle, gas exchange parameters and dry weights were determined. The ozone fumigations did not produce any effect on seedling growth. The ozone fumigation effects on gas exchange parameters were inconsistent, and generally not statistically different, with no differences occurring between seed sources. There was no correlation between photosynthetic rates and seedling growth. These results provide no evidence that ozone may be contributing to the differences in decline noted between the Mt Rogers and Mt Mitchell populations of Fraser fir.

Journal Article↗

Volatility, Adsorption, and Degradation of Propargyl Bromide as a Soil Fumigant.

During the 1960s, propargyl bromide (3-bromopropyne, C(3)H(3)Br, or 3BP) was used in a soil fumigant Trizone, a combination of chloropicrin, methyl bromide, and propargyl bromide. Since a great deal of attention is being placed on finding replacements for methyl bromide (CH(3)Br, MeBr) in soil fumigation, there is a revived interest in developing propargyl bromide as an alternative fumigant. However, at present, no information exists for assessing its behavior and safety in the environment. In this study, several important parameters needed to determine its environmental fate are obtained. These include basic parameters such as solubility, saturated vapor density, the Henry's law constant, adsorption, and degradation. These parameters are compared to MeBr and 1,3-dichloropropene (1,3-D), a methyl bromide alternative. At 25 degrees C, the Henry's law constant, K(H), for 3-bromopropyne is 0.046, which is smaller than that for MeBr and similar in magnitude to that for 1,3-D. In water at 25 degrees C, the solubility is approximately 1.5% and 3BP slowly degrades with a hydrolysis half-life of 47 days. In soil, the degradation half-life ranged from 1.2 to 5 days, depending on the soil type. Under typical agricultural conditions, 3BP will move readily in soils, a desirable characteristic for fumigants, but, due to its short soil degradation time, should not pose a serious environmental risk.

Journal Article↗

Agricultural soil fumigation as a source of atmospheric methyl bromide.

Methyl bromide (MeBr) is used increasingly as a biocidal fumigant, primarily in agricultural soils prior to planting of crops. This usage carries potential for stratospheric ozone reduction due to Br atom catalysis, depending on how much MeBr escapes from fumigated soils to the atmosphere and on details of atmospheric chemical reactions. We present direct field measurements of MeBr escape; 87% of the applied MeBr was emitted within 7 days after a commercial fumigation. Covering the field with plastic sheets retarded MeBr escape somewhat but first-day losses were still 40%; thicker sections of sheets were relatively more effective than thin sections. We also measured gaseous MeBr concentrations versus depth in the soil column; these profiles display diffusion-like evolution. In soil, MeBr is partitioned among gas, liquid, and adsorbed solid phases. Calculated soil inventories agreed only roughly with applied amounts, probably due to nonequilibrium partitioning (during the first 30 min) and to uncertainties in partitioning coefficients. Fumigated fields may release less MeBr if they are covered by more gas-tight plastic films, if injection techniques are improved and injection is deeper, and if soil moistures, organic amounts, and densities are greater than in the soil studied here.

Journal Article↗

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves.

Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K(m) for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 mum for plastidic and extraplastidic glutathione reductase, respectively, at 25 degrees C. The K(m) for NADPH was 4.8 and 4.0 mum for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L(-1) ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

Journal Article↗