Evaluation of Bioanalytical Assays for Toxicity Assessment and Mode

The toxicity of electrophiles, including reactive organochlorines, epoxides, and compounds with an activated double bond was investigated. A set of di...
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Environ. Sci. Technol. 2003, 37, 4962-4970

Evaluation of Bioanalytical Assays for Toxicity Assessment and Mode of Toxic Action Classification of Reactive Chemicals A N G E L A H A R D E R , †,‡,§ B E A T E I . E S C H E R , * ,† P A O L O L A N D I N I , †,‡ N I C O L E B . T O B L E R , †,‡ A N D R E N EÄ P . S C H W A R Z E N B A C H † , ‡ Swiss Federal Institute for Environmental Science and Technology (EAWAG), P.O. Box 611, CH-8600 Duebendorf, Switzerland, and Swiss Federal Institute of Technology (ETH), P.O. Box 611, CH-8600 Duebendorf, Switzerland.

The toxicity of electrophiles, including reactive organochlorines, epoxides, and compounds with an activated double bond was investigated. A set of different bioanalytical assays based on genetically modified Escherichia coli strains was set up to quantify cytotoxicity and specific reactivity toward the important biological nucleophiles DNA and glutathione (GSH). The significance of GSH for detoxification was assessed by cellular GSH depletion as well as by growth inhibition of a GSH-deficient strain. Tests for DNA damage comprised the measurement of induction of DNA repair systems, DNA fragmentation, and growth inhibition of a strain deficient in major DNA repair mechanisms. The most suitable assays for detection of mechanisms that underlie the observable cytotoxicity of the tested electrophiles were two sets of strains either lacking GSH or DNA repair in combination with their corresponding parent strains. Comparison of toxicity observed in those strains suggests three clearly distinguishable modes of toxic action for electrophilic chemicals: “DNA damage”, “GSH depletion-related toxicity”, and “unspecific reactivity”. The class of chemicals causing DNA damage includes the epoxides 1,2-epoxybutane, (2,3-epoxypropyl)benzene, and styrene oxide. The class of chemicals with GSH depletion-related toxicity includes compounds with an activated double bond, like acrylates and acrolein. All reactive organochlorines and some epoxides were classified as unspecifically reactive because their toxicity is initiated by reactions with both biological nucleophiles. The work presented here is a contribution for an alternative hazard and effect assessment of organic pollutants based on mode of toxic action classification.

Introduction Predictive ecotoxicological risk assessment of chemicals relies on the correct assignment of a chemical toward the relevant * Corresponding author phone: +41-1-823 5068; fax: +41-1-823 5471; e-mail: [email protected]. † EAWAG. ‡ ETH. § Present address: Swiss Federal Institute of Technology (ETH), Institute for Chemical and Bioengineering, ETH-Hoenggerberg, CH-8093 Zurich, Switzerland. 4962

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mode(s) of toxic action. A widely used approach for rough classification of chemicals into groups of different modes of toxic action is the approach of Verhaar et al., who assigned chemicals to one of four general classes based on the presence of certain functional groups (1). Chemicals were divided in narcotics, polar narcotics, reactive chemicals, and specifically acting chemicals with receptor-mediated toxicity. More detailed classification systems, based on behavioral and physiological responses of fish (2, 3) or on a set of in vitro tests (4), discriminate different types of narcotics, different types of specifically acting compounds, and reactive chemicals, which are described as either electrophilic/proelectrophilic or SH-alkylating compounds, respectively. Out of 225 arbitrarily chosen industrial organic chemicals, more than 70% were classified as narcotics, and as much as 20% were classified as reactive organic chemicals (3), indicating the industrial relevance of this chemical group. Reactive chemicals comprise substances with a large number of different reactive moieties (e.g., epoxides, isocyanates, aldehydes, or acrylates) (1). Many of these chemicals are used as intermediates in chemical synthesis or as monomers for polymerization. The economic significance is both expressed by their number and their production volume: a large number of reactive chemicals are high production volume chemicals with a yearly production volume over 1000 t (5). The production, the transport, and the use of reactive chemicals lead to a continuous emission, and particularly the transport process provides a risk for accidental releases of high amounts. As compared to baseline toxicity (i.e., the toxicity that compounds would (theoretically) have if they act only by narcosis), reactive chemicals are 10-10 000 times more toxic. In the case of electrophiles, the enhanced toxicity of reactive chemicals is presumably caused by reactions with biological nucleophilesssuch as proteins and DNAseither directly or after metabolic activation (6). Consequences of those initial molecular target interactions are toxic effects such as cell death or mutagenesis. Nevertheless, not all electrophiles react with both DNA and proteins/peptides to the same extent (e.g., for ethyl acrylate no DNA adducts were found) (7). A tendency to react preferentially with certain biological nucleophiles was observed in human health studies of occupational exposure toward reactive organic chemicals (8). In these studies, different products derived from exposure to reactive chemicals were used as biomarkers to detect human health risks. Typical biomarkers were mercapturic acids resulting from reactions with glutathione (GSH), adducts with amino acids in the main serum proteins hemoglobin and albumin, and DNA adducts, which result from alkylation of DNA bases. Van Welie et al. (8) found that exposure to ethylene oxide and 1,2-dibromoethane results in ratios of 1:10 and 1:107 of DNA to GSH adducts, respectively. On the basis of the hard and soft acids and bases (HSAB) concept, first introduced by Edwards and Pearson (9), they proposed that proportions between GSH and DNA adducts are related to the hardness of the electrophiles. Accordingly, hard electrophiles (e.g., ethylene oxide) have a higher tendency to react with the hard nucleophile DNA than soft electrophiles such as 1,2-dibromoethane; consequently, the proportion of GSH adducts is higher for soft electrophiles than for hard electrophiles. The aim of this study was to evaluate if the toxicity of electrophiles is determined by reaction with both GSH and DNA or specific reaction with either GSH or DNA. With respect to the HSAB concept, a set of reactive chemicals with different reactive moieties covering the spectrum from soft 10.1021/es034197h CCC: $25.00

 2003 American Chemical Society Published on Web 09/25/2003

FIGURE 1. Toxicokinetic and toxicodynamic effects from single cells to populations (left-hand side) and related bioanalytical assays (right-hand side) for the evaluation of single and combined toxicodynamic effects. acrylates to hard epoxides was chosen. Another important goal of this work was to find bioanalytical assays that can be used for reliable identification of reactive chemicals in addition to a set of assays indicating other toxic mechanisms (10). Combined with the prediction of internal effect concentrations, this mechanism-based mode of toxic action classification would facilitate the process of ecotoxicological risk assessment and set it on a more scientifically sound basis (11).

Design of the Bioanalytical Assay Set The set of bioanalytical assays that was tested for the evaluation of reactive chemicals allows the determination of effects, step after step, after the initial molecular interaction between the electrophile and the cellular nucleophiles (Figure 1). Included are the direct measurement of target interaction, the determination of biochemical responses for compensation of deleterious effects, the determination of physiological effects, and the quantification of population effects. Comparison of the growth inhibition of reference strains to their mutants, either lacking GSH or major DNA repair mechanisms, offers the opportunity to evaluate the relevance of the target interaction and the biochemical response on population level. A detailed description of the genetic particularities of the used strains is given in Table 1. Characteristics of the genotype that are crucial for the purposes of this investigation are detailed below. The Escherichia coli strain CC102 was used to measure growth inhibition as a general toxicity marker and mutagenicity. The detection of mutagenicity is based on the reversion of a point mutation in the gene of β-galactosidase (lacZ), which results from the transition from guanosine to adenine. As compared to strains bearing other point mutations in the lacZ gene, strain CC102 was shown to have the highest sensitivity against alkylating chemicals (12); however, CC102 does not display any deficiency of DNA repair enzymes. The detoxifying effects of GSH on population growth were evaluated using the strains MJF276 and MJF335. These two strains differ in the capability to produce GSH, which is synthesized in a two-step process. In the first step, γ-glutamylcysteine synthetase combines glutamate with cysteine; in the second step, GSH synthetase links glycine (13) to the

dipeptide to produce GSH. While mutants that are deficient in GSH synthetase (gshB) still possess a (de)toxifying dipeptide, γ-glutamycysteine synthetase mutants (gshA) totally lack the capability of (de)toxification with cysteine-containing peptides. MJF335 is a gshA mutant. Both strains MJF276 and MJF335 are additionally deficient in two potassium ion channels, which are regulated by GSH adducts (14). The efflux of potassium, and thereby destabilization of cytoplasmatic pH, was avoided by using a medium with high potassium concentration (see Experimental Section). The strains MV1161 and MV4108 were used to evaluate the importance of DNA as a target for reactive chemicals and the role played by DNA repair counteracting their toxicity. Alkylative damage of DNA can be repaired by several DNA repair pathways, which either recognize specific DNA adducts or have a broad adduct specificity. The repair processes (15) include specific transfer of small adducts, which is typical for the adaptive response (Ada), excision of specific alkylated bases by glycosylases (e.g., TagA, AlkA), and the unspecific excision of nucleotide sequences around alkylated bases, which is typical for the SOS response (e.g., RecA, UvrA). MV4108 contains a number of mutations in described DNA repair enzymes and is therefore extremely sensitive toward DNA-damaging chemicals. PQ37 and MV3766 were used to detect the induction of DNA repair processes. The measurement of the induction of DNA repair processes in these strains is based on the induction of the lacZ reporter gene, encoding the enzyme β-galactosidase. In PQ37, the lacZ gene is coupled to the promoter of the sfi gene belonging to the SOS response; in MV3766, lacZ is coupled to the promoter of the ada gene belonging to the adaptive response. Thus, induction of lacZ in PQ37 is triggered by single-strand breaks in DNA (for details of SOS response, see ref 16); while in MV3766, lacZ is induced by alkylated phosphodiesters in DNA (for details of adaptive response, see ref 17). For detection of direct target interaction either with GSH or DNA, the E. coli strain DPD2794 was used. DPD2794 carries a 17.55-kbp multicopy plasmid in which the lux genes of Vibrio fischeri are coupled to the promoter of the recA gene (18). The product of the recA gene is the control element of SOS response in bacteria. RecA binds to single-stranded DNA, which may result as a consequence of direct chemical damage VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Description of E. coli Strains strain

chromosomal and episomal genotypea

CC102

ara ∆(lac proB), F with lacI-, lacZ-

MJF276

kdpABC thi rha lacI lacZ kup KefB KefC::Tn10; Fas MJF 276, plus gshA::Tn10(Kanr)

MJF335 MV1161 MV4108

argE his ∆(gpt-proA) leu ara galK lacY mtl xyl thi rpsL supE tsx rfa; Fas MV1161, plus ∆(srlC-recA)::Tn10 uvrA ∆(ada-alkB::Camr) alkA tagA zhb::Tn5 thr rfb mgl kdgK

MV3766

as MV1161, plus lacZ::TetralkB::Tn10 (lacZ Camr)

PQ37

sfi::mud(Ap lac) cts lac∆U169 uvrA galE galY phoC rfa thr leu his pyrD thi trp::Muc srl::Tn10; F-

DPD2794

galK lac rpsL; pUCD615 (Ampr, Kanr) with recA’::luxCDABE, F-

crucial characteristics for bioanalytical assays

ref or source

inactivated chromosomal lactose operon (lac) including β-galactosidase gene lacZ, inactivated episomal lacZ can be restored by a single-point mutation capable of GSH synthesis

12

not capable of GSH synthesis (glutathione biosynthesis gene gshA inactivated by insertion of transposon Tn10) contains all DNA repair enzymes

14

following DNA repair pathways are inactivated: SOS response (recA, uvrA), adaptive response (ada, alkB, alkA), and repair of 3-methyladenine (tag) inactivated chromosomal β-galactosidase gene lacZ, episomal lacZ-reporter gene coupled to promoter of adaptive response alkB gene inactivated chromosomal lactose operon (lac) including β-galactosidase gene lacZ, insertion of lacZ-reporter gene coupled to promoter of gene sfi belonging to the SOS response luciferase reporter gene sequence (luxCDABE) coupled to the promoter of recA belonging to the SOS response

gift from M. Volkert (University of Massachusetts, Worcester, MA) P. Landini (EAWAG, Duebendorf, Switzerland) 21

14

20

18

a Abbreviations used for description of the genotype are explained on http://www.ecocyc.com (Encyclopedia of Escherichia coli Genes and Metabolism, ECOCYC 2002) (22).

to DNA or from DNA repair activities. Upon binding to singlestranded DNA, RecA is activated and triggers the proteolysis of the LexA repressor. This in turn enables the expression of an array of proteins participating in DNA repair processes. Because of the high copy number of this plasmid, the number of LexA molecules present in the cell is insufficient to bind all recA promoters and to repress its expression. Thus, even without DNA damage, high background levels of luciferase were observed (for details of the mechanism, see ref 19). Taking advantage of this phenomenon and using the reduction of light emission reflecting a reduced energy state, an unspecific toxicity end point for cell vitality was defined. Severe DNA damage by reactive chemicals was determined by plasmid DNA fragmentation. GSH depletion was determined by quantification of isolated cellular GSH.

Experimental Section Chemicals. The set of electrophiles comprised (for structures, see Figure 2) benzyl chloride (BCl, CAS Registry No. 10044-7), 3-methylbenzyl chloride (3MBCl, CAS Registry No. 62019-9), 4-nitrobenzyl chloride (NBCl, CAS Registry No. 10014-1), 2,3-dichloro-1-propene (DClP, CAS Registry No. 7888-6), trans-1,4-dichloro-2-butene (DClB, CAS Registry No. 110-57-6), styrene oxide (SOX, CAS Registry No. 96-09-3), (2,3-epoxypropyl)benzene (EPOX, CAS Registry No. 443624-2), 2-(4-nitro-phenyl)oxirane (NOX, CAS Registry No. 638874-5), 1,2-epoxybutane (EOX, CAS Registry No. 106-88-7), epichlorohydrin (EPI, CAS Registry No. 106-89-8), (1S,2S)(-)-1-phenylpropylene oxide (PPOX, CAS Registry No. 451866-5), 2-methyl-2-vinyloxirane (MVIN, CAS Registry No. 183894-4), acrolein (ACR, CAS Registry No. 107-02-8), ethyl acrylate (EA, CAS Registry No. 140-88-5), 2-hydroxyethyl acrylate (HEA, CAS Registry No. 818-61-1), isobutyl acrylate (IBA, CAS Registry No. 106-63-8), acrylonitrile (ACN, CAS Registry No. 107-13-1), and acrylamide (ACA, CAS Registry No. 79-06-1). BCl, NBCl, EPI, EOX, ACR, EA, HEA, IBA, and ACA were purchased from Fluka Chemie AG, Buchs, Switzerland. 3MBCl, DClP, and SOX were obtained from Sigma-Aldrich Chemie AG, Steinheim, Germany. DClB, NOX, EPOX, PPOX, and MVIN were bought from Aldrich Chemical Company 4964

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FIGURE 2. Structures of the investigated organochlorines, epoxides, acrylates, and acrylic compounds. Inc., Milwaukee, WI. ACN was bought from Riedel de Hae¨n, Seelze, Germany. All chemicals were of the highest purity available (g95%) and used as received; ACA was used as a 40% aqueous solution. Quantification of Growth Inhibition. All experiments with strain CC102 were performed in minimal medium (MM, pH 7.0), which consisted of 33 mM KH2PO4, 60 mM K2HPO4, 7.6 mM (NH4)2SO4, 1.7 mM sodium citrate, 1 mM MgSO4, 0.1 ‰ (w/v) vitamin B1, and 11 mM glucose as a sole carbon source. Cells were grown aerobically at 30 °C on a shaking incubator to early exponential phase (cell densities 7 × 107-1 × 108

cells/mL). Thereafter, geometrical dilution series of most chemicals were directly made with cell suspension in glass tubes closed with Teflon-coated screw caps. The solid electrophiles NBCl and NOX were dissolved in cyclohexane, and different quantities of stock solution were pipetted into the glass tubes. After the evaporation of cyclcohexane, crystallized NBCl and NOX were quickly redissolved with cell suspension. Different volumes of aqueous ACA solution were directly added to aliquots of cell suspension; differences of the resulting sample volume were corrected by adding water. Seven to eight treated samples and controls in duplicate were then incubated for a time (t) of 3 h at 30 °C on a shaking incubator. The gas volume in the closed tubes was sufficient for aerobic growth during the 3-h incubation time. Experiments were performed at least in duplicate. Growth was monitored by light scattering at 600 nm (OD600), and growth related to control was calculated according to

% growth of control ) OD600,t(sample) - OD600,t)0(control) OD600,t(control) - OD600,t)0(control)

× 100% (1)

The concentrations resulting in 50% inhibition of growth (EC50) were derived from a logistic fit of the concentrationeffect curves (eq 2), using the software Prism (GraphPad Software, San Diego, CA), which computed the best fit for experimental data of all parallels under the prerequisites of fixed minimum at 0% and fixed maximum at 100% growth. Adjustable parameters were the slope (m) and the EC50:

% effect )

100% 1 + 10

m×(log EC50 - log concn)

(2)

EC50 values of chemicals with high air-water partitioning coefficient (DClP, DClB, EA, IBA) were corrected for the loss of the chemical to the gas phase. Determination of Glutathione Influence on Growth Inhibition. The determination and calculation of growth inhibition of MJF276 and MJF335 were performed identically to CC102 (see above) but, to obtain reliable differences in growth, the incubation time with chemicals was increased to 6 h. Precultures of MJF276 contained 25 mg/L tetracycline, and precultures of MJF335 were grown with 25 mg/L kanamycine. The influence of GSH on growth inhibition of the used chemicals was characterized by the toxic ratio (TR) of EC50 values of MJF276 and MJF335 (eq 3), TRGSH. Confidence limits of the TR values were derived from error propagation from the standard deviation of the EC50 values (23):

TRGSH )

EC50 MJF276 EC50 MJF335

(3)

Determination of the Influence of DNA Repair on Decrease of Colony Forming Units. MV1161 and MV4108 at cell densities between 2 and 2.5 × 108 cells/mL were incubated with chemicals for 45 min, identical to the procedures described for CC102 (see above). MM was supplemented with 2.5% Luria-Bertani medium (LB) (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl); precultures of MV4108 were grown with 25 mg/L tetracycline. Likely due to its numerous mutations, the strain MV4108 showed a tendency to form filaments when incubated with chemicals. This made it difficult to set up a linear correlation between cell number and optical density. Instead of measuring the light scattering at 600 nm, it was necessary to evaluate the effect of chemicals on the growth on MV1161 and MV4108 by counting colony forming units (cfu). After the incubation with the chemicals, cells were spun down by centrifugation. The supernatant was removed, and the cells were resus-

pended in phosphate buffer of the same composition as the used MM. Dilution series of cells were made between 1:102 and 1:105. Aliquots of the dilution series of MV1161 were plated in duplicate on LB plates (LB medium plus 15 g/L agar) and incubated for 1 d at 30 °C before counting. MV4108 was plated in duplicate on LB plates that contained 25 mg/L tetracycline, incubated at 37 °C, and counted after 2 d of incubation. Experiments were performed only once. The percentage of the decrease of cfu of treated samples was calculated by division with the average number of cfu of the controls. EC50 values were derived from the concentrationresponse curves with eq 2. The influence of DNA repair on growth inhibition of used chemicals was characterized by the toxic ratio of EC50 values of MV1161 and MV4108 (eq 4), TRDNA:

TRDNA )

EC50 MV1161 EC50 MV4108

(4)

Induction of DNA Repair Processes. Both PQ37 and MV3766 were grown in MM with 2.5% LB on a shaking incubator to cell densities between 7 × 107 and 1 × 108 cells/ mL. Precultures of PQ37 were grown with addition of 25 mg/L tetracycline; precultures of MV3766 were supplemented with 25 mg/L chloramphenicol. The measurement of β-galactosidase induction was performed according to the method described by Miller (24) with the following modifications: isopropyl-β-D-thiogalactoside was omitted from the culture, and cells were lysed with 1 drop of 0.1% sodium dodecyl sulfate (SDS) and 2 drops of chloroform instead of toluene. Experiments were performed once in two series with overlapping concentration in order to find the concentration with maximal induction. Only samples with growth higher than 50% of control (eq 1) were used for further analysis. As positive controls, 2-methyl-2-vinyloxirane was used for the SOS response, and methyl methane-sulfonate was used for the adaptive response. Determination of Mutation Rates. CC102 was grown in MM at 30 °C on a shaking incubator to cell densities of 5-7 × 108 cells/mL. Geometrical dilution series of acrylates and acrylic compounds were directly made with cell suspension in gastight glass tubes (procedure for ACA, see above). Seven to eight treated samples and controls in duplicate were then incubated for 3 h at 30 °C on a shaking incubator. Growth was monitored by light scattering at 600 nm (OD600), and growth related to control was calculated according to eq 1. After incubation, aliquots of cell suspension were diluted 1:105 in phosphate buffer of the same composition as the used MM. Aliquots of the dilution series were plated in duplicate on LB plates and incubated for 1 d at 37 °C, and colonies formed were counted thereafter. For determination of mutants, 2-mL aliquots of the intoxicated samples were spun down and washed once with MM. The supernatant was removed, leaving approximately 100 µL that was directly plated on MM-lac plates, containing 5.8 mM lactose instead of glucose as a carbon source. The number of revertants was counted after 2 d of incubation at 37 °C. Experiments were performed only once. The mutation rate was determined by division of the number of revertants per milliliter of cell culture on MM-lac plates with the number of cfu per milliliter determined on LB plates. Only samples with growth higher than 50% of control according to eq 1 were used. Determination of Cell Vitality, Glutathione Depletion, and Detection of DNA Strand Breaks. To yield high amounts of DNA and GSH, E. coli strain DPD2794 was grown to late exponential phase with cell densities between 6 × 108 and 7 × 108 cells/mL in MM supplemented with 25 mg/L kanamycine at 30 °C on a shaking incubator. After reaching the necessary cell density, cells were incubated with chemicals as described for strain CC102 for 45 min at 30 °C. Thereafter, VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. EC50 Values Indicating Toxicity of Different E. coli Strains, EC50 Values of GSH Depletion, and Toxicity Ratios of GSH and DNA Repair-Sufficient and -Deficient Strainsa electrophile

EC50 (mM) CC102

EC50 (mM) DPD2794b

EC50 (mM) MJF276c

EC50 (mM) MJF335

ACR NBCl 3MBCl DClB BCl DClP NOX IBA HEA EA EPOX PPOX EPI SOX ACN MVIN EOX ACA

1.65 × 10-2 0.117 0.372 0.381 0.466 0.511 0.545 1.45 1.72 1.79 2.83 3.25 3.41 3.65 5.43 32.2 46.2 77.9

9.42 × 10-3 1.37 × 10-2 0.153 0.271 0.137 0.203 4.94 × 10-2 0.514 0.220 0.653 0.516 0.598 11.0 0.188 4.24 21.5 24.4 16.1

1.04 × 10-2 g 6.40 × 10-2 0.449 0.161 0.391 0.225 0.234 1.21 0.704 1.00 1.12 1.57 4.79 2.10 2.38 31.9 26.2 32.1

2.13 × 10-3 g 1.44 × 10-2 0.282 4.60 × 10-2 0.146 8.75 × 10-2 8.56 × 10-2 0.433 0.153 0.186 1.15 2.18 2.03 1.81 0.570 14.6 24.8 7.53

TRGSHd

EC50,GSH (mM) DPD 2794

EC50 (mM) MV1161e

EC50 (mM) MV4108

4.9 (2.1-11.3) 4.4 (1.8-11.1) 1.6 (0.6-4.1) 3.5 (1.2-10.2) 2.7 (1.1-6.6) 2.6 (1.2-5.7) 2.7 (1.2-6.4) 2.8 (1.1-7.4) 4.6 (1.9-11.4) 5.4 (2.4-12.1) 1.0 (0.4-2.4) 0.7 (0.4-1.4) 2.4 (1.0-5.6) 1.2 (0.6-2.2) 4.2 (1.9-9.4) 2.2 (0.9-5.4) 1.1 (0.4-2.9) 4.3 (1.8-9.9)

6.91 × 10-2 h 0.279 1.02h no depletion 1.56h 3.50h 0.234 2.85 0.882 1.68 7.18 no depletion 16.1 no depletion 6.52 95.2 114.7 44.0

ndi 0.58 0.56 1.7 1.0 2.9 2.0 j nd 8.2 nd 10 j 5.4 34 13 j 21 78 177 j 390

nd 8.5 × 10-2 4.4 × 10-2 6.8 × 10-2 0.13 0.48 0.16 nd >5.9k nd 0.34 2.1 0.36 0.26 >30k 0.27 1.4g 1146

TRDNAd 7 (1-68) 13 (1-213) 25 (7-87) 8 (0-531) 6 (0-111) 13 (3-45)