Structure-toxicity relationships for selected weak acid respiratory uncouplers

Aquatw Toxwology, 17 (1990) 239-252

239

Elsevier

AQTOX 00412

Structure-toxicity relationships for selected weak acid respiratory uncouplers M a r i e l y C a j i n a - Q u e z a d a a n d T. W a y n e Schultz* College of Veterinary Medicine, The Umversity of Tennessee, Knoxvdle, TN 37901-1071, U.S.A. (Received 3 March 1989; accepted 5 April 1990)

The relative biological response (log BR) for each of 30 select substituted phenols and amhnes was evaluated using the 48-h Tetrahymenapyriform~s population growth test system Simple linear regression analysis of log BR versus log Kow (1-octanol/water partition coefficient) was used to formulate a quantitatwe structure-activity relattonship (QSAR). The equation, log BR = 0.438 (log Kow) + 0.157; n=27, r2=0.933, s = 0 151, f=348.02, is a highly predictive model. An evaluation of data on fathead minnow mortality for these same compounds shows a similar QSAR, log LCs0= - 0 590 ( l o g / ~ ) - 3.247, n = 11, r 2= 0.917, s = 0.287, f = 99.04. These relationships are the respective QSARs for the respiratory uncouphng mechamsm of action of selected weak acids. Moreover, the toxic response m the two systems are highly correlated (r 2= 0.915) Key words: Respiratory uncouplers; structure-actwlty relationships, l-octanol/water partition coefficient;

Tetrahymenapyrtformls

INTRODUCTION

Respiratory uncouplers in the broadest sense are chemicals that elicit their effect by abolishing the coupling of substrate oxidation to ATP synthesis. However, as noted by Hanstein (1976), there are a variety of uncouplers, which include the weak acid, hydrophobic ion, and SH-reactive types. The weak acid uncouplers are the 'classic' or 'true' uncouplers and are chemicals which resemble 2,4-dinitropbenol in their action (Slater, 1966). The common structural characteristics among members of this group of uncouplers are the presence of a phenolic or anilinic moiety (i.e., a moderately weak acid on a bulky, hydrophobic aromatic ring system) and additional electron-withdrawing substituents. Such uncouplers have pi electrons, which delocalize the charge on the anions and increase the solubility of the anions in the low Correspondenceto" T.W. Schultz, College of Veterinary Medicine, The Umverslty of Tennessee, Knoxville, TN 37901-1071, U S.A 0166-445X/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

240

dielectric interior of lipid bilayers. Since such chemicals are known to increase H + transfer across membranes (Mitchell, 1966), they are also termed proton ionophores or protonophores. The classic weak acid protonophore, 2,4-dinitrophenol, was shown by Loomis and Lippman (1948) to uncouple electron transport and ATP synthesis in mitochondria. This led to a variety of investigations during the 1960s and 1970s into the action of uncouplers (for reviews see McLaughlin and Dilger, 1980 and Terada, 1981). An examination of the literature reveals that several structure-activity relationship investigations of various respiratory uncouplers have been conducted over the past 20 years. These include the studies of Stockdale and Selwyn (1971), Tollenaere (1973) and Miyoshi et al. (1987) with phenols, B~ichel and Draber (1974) with hydrazones, Terada et al. (1974) with fenamic acids, Labbe-Bois et al. (1975) with dicoumarols, and Terada et al. (1988) with salicylanilides. Schultz and co-workers compared the relative toxicity and the l-octanol/water partition coefficient (log Kow) dependent quantitative structure-activity relationships (QSAR) for a heterogeneous series of phenols. This study included tests on 2,4-dinitrophenol, pentachlorophenol and several other weak acid protonophores (Schultz et al., 1986). They used data generated for the Pimephales promelas 96-h mortality assay and the 48-h Tetrahymena population growth assay and demonstrated several points: (A) phenols elicited a toxic response by one of two mechanisms of action, polar narcosis or the uncoupling of oxidative phosphorylation (i.e., respiratory uncouphng); (B) a linear log Kow dependent QSAR could be developed for each mode of action in both test systems; (C) the toxic response in one system could be used to accurately predict the response in the other system; and (D) the presence of two nitro substituents or four or five halogen substituents is correlated with the uncoupling mode of action (Schultz et al. 1986). These findings were corroborated by McKim et al. (1987), who exposed spinally transected rainbow trout to 2,4-dinitrophenol and pentachlorophenol to define the fish acute toxicity syndrome (FATS) for respiratory uncouplers based on a series of physiological-biochemical parameters. It became clear that the weak acid uncoupling of oxidative phosphorylation was a distinct mechanism of toxic action which could be related to a specific molecular structure and defined by a set of whole animal respiratory-cardiovascular responses. Veith and Broderius (1987), noted that the fish mortality QSAR for the type (II) narcosis syndrome (i.e., polar narcosis) was based on the toxic response for selected anilines, as well as phenols. Of equal importance was the fact that the relative toxicity reported for other anilines and phenols, including dinitro-, polyhalogen- and phenylazo-derivatives, did not model well by either the nonpolar narcosis (Veith et al., 1983) or polar narcosis (Veith and Broderius, 1987) QSAR. Efforts in this laboratory have expanded the Tetrahymena QSAR for polar narcotics to include a variety of monosubstituted phenols (Schultz, 1987a), as well as selected alkylated or halogenated anilines (Schultz et al. 1989). Recently a Tetrahymena QSAR was developed for nonpolar narcotics (Schultz et al., 1988).

241 We hypothesized that dinitro-, tetra- and pentahalogen-, and phenylazo-substituted anilines act as weak acid protonophores or uncouplers. Moreover, we further hypothesized that dihalogen-mononitro-substituted anilines and phenols act in the same manner. Since compounds eliciting their toxic response via the same mode of action should be modeled by the same QSAR, it was likely that a single log Kow dependent QSAR could be generated which would accurately model the toxicity of all of these chemicals. In an effort to explore this possibility, we have determined the relative toxicity of each of a series of select substituted anilines and phenols, using the Tetrahymenapopulation growth impairment assay. We have also evaluated these chemicals for aquatic persistence and explored possible correlations between the toxicity of the two systems and log Kow. In addition, we have compared the observed and predicted toxicity of other potential weak acid uncouplers with varying molecular structure. MATERIALSAND METHODS The Tetrahymenapyriformis test system was used under static conditions. This bioassay uses population densities of axenic cultures as its endpoint. Each chemical was tested in duplicate for a minimum of three replicates following initial range finding tests. The ciliate was reared in axenic culture with population levels measured spectrophotometrically as absorbance at 540 nm following 48-h of incubation. Each replicate was at minimum a five-step graded concentration series using freshly prepared stock solutions. Assays were conducted in 250 ml Erlenmeyer flasks containing 50 ml of proteose peptone-based medium (Schultz, 1983). Cultures without test chemical were used as controls. Only tests with control absorbances between 0.6 and 0.9, late log phase growth, were used in the evaluations. The test chemicals examined in these investigations, all potential weak acid protonophores, were selected based on several criteria. All were available commercially (Aldrich Chemical Company, Milwaukee, Wisconsin, U.S.A.; Fluka Chemical Corporation, Ronkonkoma, New York, U.S.A.) in sufficient purity (95% or greater) so as not to have to be repurified prior to testing. In addition, all were aniline or phenol derivatives with a phenylazo substitution, two nitro substitutions, four or five halogen (i.e., F, CI, Br or I) substitutions, or one nitro and two halogen substitutions. Stock solutions were prepared in dimethyl sulfoxide (DMSO) at concentrations of 5, 10, or 25 g/l. In every case, the volume of stock solution added to each flask was limited so the final DMSO concentration did not exceed 0.75 %, an amount which does not alter Tetrahymenareproduction (Schultz and Cajina-Quezada, 1982). Population growth inhibitions were computed with the probit procedure of Statistical Analysis System (SAS). The IGCs0 (50 % inhibitory growth concentration) and the 95 % fiducial interval were determined for each tested chemical by measuring the present control-normalized absorbance (Y) and the toxicant concentration in mg/l (X). For comparative purposes, acute toxicity data for fathead minnows (log LCs0)

242 tested with a standard flow-through protocol (USEPA, 1975) were collected on select chemicals from the four volume series Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas). A QSAR was developed using log BR (biological response), the log of the inverse of the IGCs0 in mmol/1, as the dependent variable and log Kow as the independent variable. These data were modeled using simple regression analysis (general linear model procedure of SAS). Model adequacy was measured by the coefficient of determination (r2). In addition, the square root of the mean square for error (s) and ratio of model mean square to the error mean square (f) were noted, as was the level of significance ( P , > f ) of the molecular descriptor. Similarly, log LCs0 was regressed against log Kow. These log Kow values were computed calculated by the Pomona College Medicinal Chemistry Project CLOGP version 3.34 software or retrieved as measured values from the select list for comparison in the same program. In addition, log LCs0 was regressed against log BR. Chemical persistence studies were undertaken for each test chemical with the aid of HPLC. In each case, the chemical was analyzed twice. The analyzed solutions consisted of the appropriate aliquot of stock added to 50 ml of sterile distilled water in a foam stoppered 250-ml Erlenmeyer flask, so as to make the final concentration of the test chemical approximately equal to that of its IGCs0 value. For analysis, a Waters Model 840 HPLC with a C18 reverse-phase column, with a Waters Model 600 Multisolvent Delivery System and a Model 712 Waters Intelligent Sample Processor were used. The test chemicals were eluted using a degassed 65/35 mixture of methanol and 0.5 M ammonium acetate buffer adjusted to a pH of 6.7. The solvent flow rate was set at 1.0 ml/min. The absorbance detector, set at 254 nm, recorded directly onto a Digital Professional 350 microprocessor. At t = 0 h, a 10-20/tl aliquot was injected into the HPLC and eluted for 4 to 10 min, depending on the derivative. Peaks were integrated using Waters Expert Software. The sample-containing flasks were then placed under the same environmental conditions as those for the bioassay. At t = 48 h, the test solutions were again injected onto the HPLC and analyzed using the same method. Percent loss was measured as the difference between the t = 0 and t = 48 concentration. Several other chemicals, including a number of known lipophilic weak acid uncoupling agents, were also tested and their observed and predicted toxicity compared. RESULTS Table I lists the Chemical Abstract Services (CAS) registry number and aquatic persistence data for each test chemical. All chemicals tested show minimal abiotic loss (i.e., less than 2.5 %). The log BR, log LCs0 and log Kow values used in these analyses are presented in Table II. A scatter plot of log BR versus log Kow is shown in Fig. 1. Least-squares linear regression analysis of these data resulted in the equation:

243

[1]

Log BR = 0.438 (log Kow) + 0.157 n=27, r2=0.933, s = 0.151,f= 348.02. I n E q n . 1, l o g Kow is a h i g h l y significant p r e d i c t o r o f t o x i c i t y ( P r > F = 0 . 0 0 0 1 ;

df

1, 25). A s c a t t e r p l o t o f l o g LCs0 v e r s u s l o g Kow is s h o w n in Fig. 2. R e g r e s s i o n a n a l y s i s o f these d a t a r e s u l t e d in t h e e q u a t i o n : TABLE I AQUATIC PERSISTENCE OF SELECT WEAK ACID UNCOUPLERS No

1

2 3 4 5. 6 7 8 9. I0 11

12 13. 14 15 16 17 18. 19. 20. 2 I. 22. 23 24 25 26 27 28 29 30

Compound

CASa number

Percentage loss 4-b standard deviation

4-phenylazophenol 2,4-dtmtrophenol 2,5,dim(rophexaol 2,6-dimtrophenol 2,5-dtmtro- l-naphthol 2;6.d~ltro-4-m~hylphenol 4~6-dmlt ro-2-metl~ytphenol 2,6-d~bromo-4-naropbenol 2,44-~ichloro-6-mtrophenol 2~6-dnodo-4-mtrophenol 2,~,4,5-tetrachloropbenol 2.,3,5,6-tetrachloropbenol 2~3,5,6-tetrafluorophenol 3,4,5,6-tetrabromo-2-methylphenol pentabromophenol pentachlorophenol pentafluorophenol 4-phenylazoandme 2,4-dmltroandme 2,6-dmltroanlllne 3,5-dmltroamhne 2,6-dmltro-4-methylanlhne 2,4-dlbromo-6-mtroamhne 2,6-dlbromo-4-nltroamhne 2,4-dlchloro-6-mtroanlhne 2,6-dlchloro-4-mtroanlhne 4,5-dlchloro-2-nltroamhne 4,5-dlfluoro-2-nltroamllne 2,3,4,5-tetrachloroamhne 2,3,5,6-tetrachloroandme

1689-82-3 51-28-5 329-71-5 573-56-8 605-69-6 609-93-8 534-52-1 99-28-5 609-89-2 305-85-1 4901-51-3 935-95-5 769-39-1 576-55-6 608-71-9 87-86-5 77 I-61-9 60-09-3 97-02-9 9606-22-4 618-87-1 6393-42-6 827-23-6 827-94-1 2683-43-4 99-30-9 6641-64-1 78056-39-0 634-83-3 3481-20-7

0 0 0 0 0 2.20_+ 220 0 0 0 0 0 0 1 704-1.70 0 0.50_ 0.50 2.45 + 2 45 2.50 + 2.50 0 0 0 0 NT c 0 NT c 0 NT ¢ 0 0 0 0

~Chemlcal Abstracts Services registry number bpercentage loss based on a 48-h period cNT, not tested

244 T A B L E II SUMMARY OF TOXICITY AND MOLECULAR DESCRIPTORS FOR SELECTED WEAK ACID UNCOUPLERS No.

Compound

Log BR

Log Ko,,a

Log LCs0-i b

I. 2. 3. 4 5 6. 7 8. 9 10 11 12. 13. 14 15. 16 17 18. 19 20. 21 22. 23 24 25. 26 27 28 29 30.

4-phenylazophenol 2,4-dimtrophenol 2,5-dimtrophenol 2,6-dinitrophenol 2,5-dimtro-l-naphthol 2,6-&mtro-4-methylphenol 4,6-dimtro-2-methylphenol 2,6-dibromo-4-mtrophenol 2,4-diehloro-6-nitrophenol 2,6-dnodo-4-mtrophenol 2,3,4,5-tetrachlorophenol 2,3,5,6-tetrachlorophenol 2,3,5,6-tetrafluorophenol 3,4,5,6-tetrabromo-2-methylphenol Pentabromophenol Pentachlorophenol Pentafluorophenol 4-phenylazoamhne 2,4-dlmtroanihne 2,6-dmltroanihne 3,5-dmltroanlhne 2,6-&mtro-4-methylandme 2,4-&bromo-6-mtroaniline 2,6-dlbromo-4-nitroanihne 2,4-dlchloro-6-mtroamline 2,6-dlchloro-4-mtroanihne 4,5-dmhloro-2-nitroamhne 4,5-dlfluoro-2-nltroamhne 2,3,4,5-tetrachloroamhne 2,3,5,6-tetrachloroandme

1.655 1.096 0 929 0.573 1.585 1 230 1.329 1.357 1.750 1.812 2 004 2.219 1 167 2 573 2.664 2 568 1.631 1.421 0.716 0.941 0.941 NESc 1 170 NESc 1.170 NES ¢ 1.714 0.764 1 956 1.762

3.18 1 54 1.75 1.22 3.09 2 47 2.56 3.05 3.21 3 76 4.33 4.31 2 07 5.70 5 69 5.12 3.23 2.62 1 84 1 37 1.37 2.47 3 52 2 78 3.22 2 34 3.22 2.08 4 33 4.31

-5.23 -4.22 -4.74 -3.57 -4.84 - 5.11 - 5 75 - 6 72 - 5.99 - 4 07 - 5 92

aComputer calculated by C L O G P version 3.34 software or retneved as measured values bRetneved from Acute Toxlcatles o f Organic Chemicals to Fathead Minnows (Pzmephalespromelas) Volumes I, II, III, and IV oNES, no effect at saturatmn.

L o g L C s o = - 0 . 5 9 0 ( l o g Kow) - 3 . 2 4 7 n = 11, r2 = 0 . 9 1 7 ,

s=0.287,f=

I n E q n , 2, l o g Kow is a h i g h l y s i g n i f i c a n t p r e d i c t o r

1, 9).

[2]

99.04. of toxicity (Pr > F= 0.0001; df

245

2 50.

2 O0

rr m

1 50

1 O0

0 50

1'0

'

2'0

'

3'0

'

4'0

'

5'0

'

log Kow

Fig. 1 A scatter plot of the log BR vs. log Kow for weak acid respnratory uncouplers One hidden value.

A scatter plot of log LCs0 versus log BR is presented in Fig. 3. Regression analysis of these data resulted in the equation:

[31

Log LCs0 = - 1.254 (log BR) - 3 . 1 3 3 n = 1 I, r2=0.915, s = 0 . 2 9 0 , f = 96.62.

This third equation revealed the toxic response in these two systems to be highly correlated. -3 6 -

*4 2

-4 8

--° ~

-5 4 .

-60.

-6 6,

'0

2'0

'

3'0

'

4'0

'

5'0

'

log Kow

Fig. 2 A scatter plot of the log LCs0 vs log Ko. for weak acid respiratory uncouplers

246 -3 6

-4 2

-4 8

-5 4.

-6 o-

-6 6

0'4

0'8

1 '2

1 '6 lOg BR

2'0

2'4

Fig 3 A scatter plot of the log LCs0 vs. log BR for weak acid respiratory uncouplers.

Table III lists the Chemical Abstracts Services (CAS) registry number, observed toxicity, predicted toxicity (based on Eqn. 1) and residual values for other potential lipophilic weak acid uncouplers. Dicoumarol (3,3'-methylene bis(4-hydroxy-2H-1benzopyran-2-one) is a blood anticoagulant and a 'classic' uncoupler (Heytler, 1979). Mefenamic acid (N-(2',Y-dimethylphenyl)anthranilic acid) is also a known uncoupler (Terada et al., 1974). Picric acid (2,4,6-trinitrophenol) is a homolog of 2,4dinitrophenol. While the observed toxicity' of dicoumarol and mefenamic acid is modeled by Eqn. 1, the toxicity of picric acid is not. DISCUSSION

Weak acid uncouplers abolish the link between substrate oxidation and ATP synT A B L E Ill TOXICITY OF OTHER POTENTIAL WEAK ACID UNCOUPLERS

Compound

CAS a number

Log Kow

Observed log BR

Predicted b log BR

Residual value

Dicoumarol Mefenamlc acid Plcric acid

66-76-2 61-68-7 88-89-1

3.40 2.37c 1.82

1.697 1 518 - 0 155

1.646 1.195 0.954

0.051 0.323 - 1,109

aChemlcal Abstract Service registry number bBased on Eqn. 1, ¢Calculated for pH 7 0 (from Hansch and Leo, 1979).

247

thesis without affecting the electron-transport mediators, although they increase the rate of electron transfer (Terada, 1981). The latter results in a decrease in the phosphorylation of ADP to ATP in relation to the amount of oxygen consumed. While it is generally accepted that weak acid uncouplers are H + conductors, the precise mechanism of proton conduction is still in question. One mechanism by which such chemicals may uncouple oxidation from phosphorylation can be explained by the chemiosmotic hypothesis (Mitchell, 1966). The chemiosmotic hypothesis predicts that a H + gradient is formed across the inner mitochondrial membrane as a result of the electron transport induced accumulation of protons. This gradient results in the movement of H + through the inner membrane, by way of the membrane bound ATP-synthetase enzyme complex and the generation of ATP. Weak acid uncouplers, like 2,4-dinitrophenol, are thought to stop mitochondrial ATP synthesis without slowing 02 uptake. Upon exposure to such an uncoupler, electron transport continues at a rapid rate, but no H + gradient is generated. The uncoupler acts as a protonophore and provided an alternative to ATP-synthetase. For this to take place, the uncoupler must be hydrophobic enough to reach and reside in the inner mitochondrial membrane. The higher pH of the matrix space causes a proton to dissociate from the weak acid moiety of the uncoupler, while the low pH of the intermembrane space causes a proton to bind to the weak acid moiety of the uncoupler. This interaction results in 'short-circuiting' and dissipation of the electrochemical proton gradient. McLaughlin and Dilger (1980) subdivided weak acid protonophores into two classes based on the mechanism by which they transport protons across membranes. In one mechanism, the H + ions are transported across the membrane by the anionic form of the weak acid. This group of uncouplers includes the substituted carbonylcyanide phenylhydrazones. In the second mechanism, the hydrogen ions are transported across the membrane by the dimeric form of the weak acid and the undissociated acid. The latter group of uncouplers includes 2,4-dinitrophenol, and pentachlorophenol (McLaughlin and Dilger, 1980). Early structure-activity studies found, especially of phenols, (A) uncoupling potency to be related to hydrophobicity measures by log Kow or its substituent constant ;r and (B) that the acid dissociation constant, pKa, or the Hammett electronic substituent constant, tr, may or may not play a role in modulating potency (Stockdale and Selwyn, 1971; Tollenaere, 1973; Labbe-Bois et al., 1975; Miyoshi et aL, 1987). Regardless of the specific of the mechanism of uncoupling it must involve the agent getting from the aqueous environment to the site of action. Therefore, hydrophobic partitioning will be an important parameter in modeling such activity. However, the significance of either ionization or electronic parameters will vary depending on the agents involved in model formulation. For example studies with isolated rat liver mitochondria revealed that the relationship between uncoupling activity and a while linear for halogenated phenols including the pentachloro-derivative was different than that for nitrophenols including the 2,4-dinitro-derivative (Stockdale and Sel-

248 wyn, 1971). Furthermore, least-squares regression analysis of the combined data showed activity to be related to both hydrophobic and electronic effects, log uncoupling -1 =0.84 n + 1.13 ~r + 2.15 with n=23 and r=0.982 (Stockdale and Selwyn, 1971). The latter relationship included not only classic weak acid uncouplers such as 2,4-dinitrophenol and pentachlorophenol but also phenol and its monomethyl, monochloro and mononitro derivatives, chemicals which recently have been considered to act as polar narcotics (Schultz et al., 1986; Schultz, 1987a, 1987b; Veith and Broderius, 1987; Schultz et al., 1988; Bradbury et al., 1989). As noted by McKim et al. (1987), the toxic response to weak acid respiratory uncouplers can be defined in spinally transected rainbow trout by a FATS (fish acute toxicity syndrome). The most dramatic physiological-biochemical responses associated with exposure to such uncouplers are the rapid and continuous increases in ventilation volume and oxygen consumption, which correspond to a continuous increase in metabolic rate (McKim et al., 1987). Recently, Bradbury and co-workers have defined a FATS for polar narcotics (Bradbury et al., 1989). The most dramatic physiological-biochemical responses associated with exposure to polar narcotics are tremors, initiated by a cough, that progress to seizures and an alteration in blood chemistry attributed to anaerobic metabolism (Bradbury et al., 1989). A review of the literature to include the investigations of Schultz et al. (1986) and Veith and Broderius (1987) coupled with the analyses presented here, show that at least for fish and protozoans anilines, like phenols, invoke their toxic response by at least two mechanisms of action: polar narcosis and the weak acid uncoupling of oxidative phosphorylation. The n- and a-dependent relationship developed by Stockdale and Selwyn (1971) models the relative toxicity of chemicals eliciting at least two models of action, uncoupling and polar narcosis. Similarly the more recent study of Ravanel et al. (1989) with chlorophenol also models chemicals thought to have two modes of action. In contrast all the chemicals modeled in the present study elicit their toxic response in the same manner as 2,4-dinitrophenol and pentachlorophenol, weak acid respiratory uncoupling. This conclusion is based on two lines of reasoning. First, each chemical possesses the structural characteristic of the classic weak acid uncouplers (Terada, 1981), each having (A) an acid-dissociable group (hydroxy or amino substituent; (B) a strong electron-withdrawing moiety (nitro and/or halogen substituents); and (C) a bulky hydrophobic group (typically a benzene ring). Second, all are modeled accurately by a unique linear log Kow dependent QSAR, Eq. [1]. As noted by Veith et al. (1984) and McKim et al. (1987) such simple QSARs can be developed for each mode of toxic action. The toxicity of other known weak acid uncouplers, such as dicoumarol and mefenamic acid, model adequately by Eqn. 1. However, the toxicity of picric acid is lower, by more than one log unit, than predicted by Eqn. 1. In fact, picric acid is best modeled as a nonpolar narcotic, fitting Eqn. 11, log BR = 0.8222 (log Kow) - 1.7889, of Schultz et al. (1988).

249

The latter may be due to steric hindrance between the two ortho-position nitro groups and the polar hydroxy moiety. However, this seems unlikely in light of the fact a variety of uncoupler actions are regulated mainly by hydrophobic and electron properties (Terida, 1981). Heytler (1979) noted a pKa range for 'classic' uncouplers is from about 4 to 8. Similarly, Terada (1981) reports a pKa range of from 5 to 7 for lipophilic weak acid uncouplers. Both of these ranges are consistent with the observations of Schultz (1987b) who suggested that pKa values were useful in determining when a phenol acted as a polar narcotic or weak acid uncoupler. The pKa of picric acid is 0.42 (Dean, 1985). This is far below the reported ranges. It may be that picric acid does not elicit its toxic response in the same manner as the other chemicals tested because it is not ionized at pH values found in the mitochondria, and there for does not meet the requirements of a weak acid uncoupler (Terada, 1981). The chemicals analyzed in these studies, while limited, do encompass an array of commercially available industrial organic chemicals. Each of which a priori was hypothesized to act as weak acid uncouplers because it meet specific structural criteria. Several conclusions may be drawn. These include: (A) excellent log Kow dependent QSARs for selected phenols and anilines can be formulated for both the Tetrahymena growth impairment and fathead minnow acute lethality endpoints (i.e., Eqns. 1 and 2); (B) both of these QSARs model phenylazo, dinitro, tetrahalogen, pentahalogen and dihalogen-mononitro derivatives of aniline and phenol; and (C) these compounds elicit their toxic response by way of the weak acid uncoupling of oxidative phosphorylation. ACKNOWLEDGMENT

This study was supported in part by The University of Tennessee's Center of Excellence in Livestock Diseases and Human Health. REFERENCES Btichel, K.H. and W. Dreber, 1974. Structure-activity correlations of acanc~dal hydrazones; uncouplers ofoxidatwe phosphorylatlon. Adv Chem. Set. 114, 141-154. Center for Lake Superior Environmental Studies, Umversity ofWisconsm -Supenor, 1984 Acute Tox~c~ties of Orgamc Chemicals to Fathead Minnows (Plmephalespromelas) Vol I , L.T. Brooke, D.J Call, D L. Gelger, and C E. Northcott (eds.). Superior, WI, 414 pp. Center for Lake Superior Environmental Studies, Umversity of Wisconsin - Superior, 1985. Acute Toxlclties of Orgamc Chemicals to Fathead Minnows (Pimephalespromelas) Vol. II., D.L. Gelger, C.E. Northcott, D.J. Call, and L T. Brooke (eds.), Supenor, WI, 326 pp. Center for Lake Superior Environmental Stu&es, Umversity of Wisconsin - Superior, 1986. Acute Toxicities of Organic Chemicals to Fathead Minnows (Ptmephalespromelas) Vol. III D L. Geiger, S.H. Poiher, L.T. Brooke, D.J. Call (eds.). Superior, WI, 328 pp Center for Lake Superior Environmental Studies, University of Wisconsm -Supenor, 1988 Acute Toxlcities of Orgamc Chemicals to Fathead Minnows (Ptmephalespromelas) Vol IV. D L Geiger, D J. Call, and L T. Brooke (eds) Superior, WI, 355 pp.

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