Uptake and biotransformation of quinoline by rainbow trout

Aquatic Toxicology, 7 (1985) 221-239 Elsevier

221

AQT 00176

UPTAKE AND BIOTRANSFORMATION OF QUINOLINE BY RAINBOW TROUT

ROGER M. BEAN, DENNIS D. DAUBLE, BERTA L. THOMAS, R. WILLIAM HANF, JR. and EDWARD K. CHESS Pacific Northwest Laboratory, P.O. Box 999, Richland, WA 99352, U.S.A. (Received 17 June 1985; revised version received 14 August 1985; accepted 26 August 1985)

Rainbow trout (Salmo gairdnerO readily absorb and metabolize [~+C]quinolinewhen exposed to l mg/I concentration in water. Juvenile fish accumulated over 60% of the total radiolabel body burden in the gall bladder as quinoline metabolites after 48 h exposure followed by 24 h in clean water. Hydrolysis products of the metabolites, isolated by alkaline digestion and base-catalysed acetylation, were found to be hydroxyquinolines and quinolinethiols. There is evidence that the hydroxy form was present in the gall bladders as the glucuronide was obtained from thin layer chromatographic experiments, and by cleavage with ~-glucuronidase. The thiols, identified by high and low resolution mass spectrometry, predominated over the hydroxy derivative in most tissues examined. Relative body burdens of quinoline plus metabolites after 48 h were in the order gall bladder > muscle > gut > eyes > liver = gill ~ kidney. Key words: rainbow trout; quinoline; metabolites; hydroxyquinoline; quinolinethiol

INTRODUCTION A q u a t i c o r g a n i s m s have the capacity, t h r o u g h a variety o f metabolic processes, to m o d i f y chemical properties o f xenobiotics taken up from the e n v i r o n m e n t . Specific e n z y m a t i c reactions that metabolize toxic c o m p o u n d s have been suggested or described for m a n y fish species (Buhler a n d Rasmussen, 1968; Pedersen et al., 1976; R o u b a l et at., 1977; Varanasi a n d G m u r , 1980, 1981; G r u g e r et al., 1981). Several o f the b i o t r a n s f o r m a t i o n reactions k n o w n to occur in fish in vivo have been described previously in m a m m a l s . In fish, b i o t r a n s f o r m a t i o n processes involve one a n d two electron oxidations, h y d r o x y l a t i o n , a n d / o r c o n j u g a t i o n o f a c o m p o u n d to polar or water-soluble forms that are m o r e easily excreted (reviewed in Lech a n d Bend, 1980). In some cases, resultant metabolites m a y be m o r e biologically active or more persistent in fish tissue t h a n the p a r e n t c o m p o u n d (Lech, 1974; G r u g e r et al., 1981; G m u r a n d Varanasi, 1982). F o r example, m e t a b o l i s m in fish may activate the toxicity of specific polycyclic a r o m a t i c h y d r o c a r b o n s ( P A H s ) by c o n v e r t i n g them to P A H epoxides that m a y b i n d to m a c r o m o l e c u l a r material and increase carcinogenicity

222 (Varanasi and Gmur, 1980). Thus, metabolism may not necessarily result in detoxification or removal of a toxic constituent from an environmental pathway. Potential for contamination of aquatic ecosystems by organic components associated with conversion of combustion or basic energy-related materials (e.g., coal, petroleum, shale oil) has prompted concerns of human health hazards via aquatic food chains. Transfer to man by important food organisms such as fish could occur for aquatic systems that serve as sinks for environmental contaminants released from energy processes. Knowledge of the fate of these materials in fish is important in evaluating the potential for such transfer. We conducted studies to examine the bioaccumulation and metabolism of the nitrogen heterocycle, quinoline, in rainbow trout (Salmo gairdneri). Quinoline is a common constituent of petroleum products and potential liquid fuel substitutes, including shale oil and coal liquids. This compound is relatively soluble in water and has been identified in a number of industrial products (Weisenberg and Williams, 1980). In addition, polycyclic nitrogen heterocycles, including quinolines, are released in effluents of coking processes (Bark et al., 1972). Some members of this chemical class are mutagenic (Guerin et al., 1980; Later et.al., 1982), and quinoline itself is a hepatocarcinogen in mice (Hirao et al., 1976). Studies of the specific mechanisms of the biotransformation of nitrogen-containing polycyclic aromatic compounds in fish are important in light of recent evidence by Roubal and Malins (1985) that indicates a causal link between free radical derivatives of nitrogen heterocycles and hepatic lesions in English sole (Parophrys vetulus). Our experiments were designed to determine the distribution and fate of quinoline within freshwater fish commonly included in the human diet. Of further interest was to investigate biotransformation pathways through characterization of metabolites accumulated in various tissues. Rainbow trout were exposed to subacute levels of quinoline in defined water systems, and tissue concentrations were analyzed by capillary gas chromatography using a nitrogen/phosphorous detector or by gas chromatography/mass spectrometry. MATERIALSAND METHODS

Materials [14ClQuinoline, uniformly labeled in the benzo ring, was synthesized from uniformly labeled benzene by Pathfinder Laboratories, Inc., St. Louis, MO, and was purified (> 99°7o) by liquid chromatography. Specific activity was 7.6 #Ci/#M. Quinoline and 5-hydroxyquinoline standards (99o70) were obtained from Aldrich. 8-Hydroxyquinoline glucuronide, 8-hydroxyquinoline sulfate, aryl sulfatase (type H-I from Helix pomatia), and fl-glucuronidase (type H-3 from H. pomatia) were obtained from Sigma Chemical Company. The/~-glucuronidase contained sulfatase activity, and the aryl sulfatase contained fl-glucuronidase activity. Acetic anhydride (Eastman) was distilled before use and stored over Drierite®. All solvents were Bur-

223 dick and Jackson 'distilled in glass' grade. NCS tissue solubilizer and PCS liquid scintillation cocktail were obtained from Amersham/Searle, Arlington Heights, IL. Bioaecumulation studies Rainbow trout 'swimup' fry, Kamloops strain, ranging from 0.21 to 0.41 g (mean size, 0.30 g) were used for determination of uptake and elimination kinetics during short-term static exposures. Fish (n = 4) were exposed in 600-ml capacity glass beakers containing 500 ml of well water. The fish were fed 24 h prior to the test but not during exposure. Temperature was maintained at 12°C by a water bath, and the light:dark cycle was 16:8 h. Acute exposures o f the rainbow trout to a range o f quinoline concentrations were conducted to estimate acceptable concentrations for bioaccumulation studies (four replicate beakers at each of seven test concentrations, n = 16 per treatment). A 48-h LCs0 o f 10.7 mg/1 quinoline was determined by the graphical method (APHA,

1981). Mean concentrations of quinoline during the 48-h uptake phase of the bioaccumulation study were 1.11 mg/l total quinoline with 1.2 t~g/l as radiolabeled quinoline. A single fish was removed from each of four randomly selected beakers at various times during the 48-h uptake and subsequent 24-h elimination phase for determination o f radiolabel concentrations. The entire fish was solubilized in 2 ml o f LCS tissue solubilizer at 40°C and sonicated to aid solubilization. The remaining fish from each beaker were frozen at - 70°C and saved for analysis o f parent compound and metabolites. Water was analyzed for total quinoline by liquid chromatography (60°70 methanol/40% water, isocratic flow over 3.9 mm x 30 cm Waters # Bondapak ® analytical column), and for radioactivity by liquid scintillation at each sample interval. Tissue distribution studies Juvenile rainbow trout used in tissue distribution and metabolism studies averaged 101 + 7 mm fork length and 10.7 ___ 2.1 g. For exposures, four fish each were held without food within 12 glass aquaria that contained 20 I of well water. Tests were conducted under static conditions o f 12°C with a light:dark cycle of 16:8 h. Aquaria were not aerated, and water was exchanged at 48 h for fish that were depurated. Initial concentrations of quinoline in the water were 1.01 + 0.07 mg/l with 1.I /~g/l as radiolabeled quinoline. One fish was removed from each of four replicate aquaria and dissected at three time intervals: 24 and 48 h after exposure, and 72 h after a 24-h depuration period. The fish were analyzed for a total ~4C activity in gall bladder plus bile, liver, kidney, gills, muscle, gut, eyes, and carcass (remaining tissue). The remaining three fish from each aquaria were also dissected at corresponding sample intervals, and their tissues were stored in pre-weighed glass vials at - 7 0 ° C for metabolite determination. All tissues were weighed (wet weight) im-

224

mediately after dissection and solubilized in 1 to 2 ml of LCS tissue solubilizer at 40°C. Only one eye was sampled, and subsamples ( - 0 . 5 g) were taken from the muscle and carcass. The carcass was weighed, blended into a puree, and the radioactivity of an aliquot was measured. Water samples (n = 4) were taken for analysis of quinoline and total radioactivity at each of the three sample intervals.

Quantitative analysis for quinoline and metabolite hydrolysis products Analysis for quinoline in water used for trout exposure was performed by reverse phase liquid chromatography. Samples of water were injected with a Waters 710B sampler onto a Bondapak ® C-18 column, which was diluted with 1 ml/min methanol (60°70)/0.1 n acetate buffer (40%). The UV detector signal (Spectroflow 773) was quantified with a Hewlett/Packard 3390A integrator. Analysis of tissue samples for quinoline, quinolinethiols, and hydroxyquinolines was conducted by digestion with alkali, followed by formation of the acetate derivatives and quantification by gas chromatography. Tissue samples (2 g or less) were treated with 1 ml 5 N NaOH at 90°C for 1 h in Teflon®-capped scintillation vials. After approximately 10o70 of the sample was subsampled for scintillation counting, 2 ml n-heptane, 10 mg Na2CO3, and 0.5 ml acetic anhydride were added to the vials. After shaking and heating to 80°C, Na2CO3 was then slowly added over a period of 15 min to neutralize excess acetic acid. Addition was continued until CO2 evolution ceased. The heptane layer was removed after the mixture was shaken for 1 rain and heated to 80°C to break emulsions. The sample was extracted with an additional 2 ml heptane, and the combined hexane extracts were extracted three times with 0.5 ml 0.1 N HCI. (Caution: Heating the sample at this point to separate layers hydrolyzes the acetate derivatives; emulsions should be separated by centrifuge). The HCI extracts were neutralized with NazCO3, and extracted with 1 ml n-heptane, followed by 1 ml n-hexane. Two/zl of a 2 mg/ml solution of 3-methylindole was added to the combined extracts to provide an internal standard. The sample volume was brought to 2.0 ml, and a 0.2 ml aliquot was removed for scintillation counting. Samples were evaporated under a stream of nitrogen to 0.3 ml for gas chromatographic (GC) analysis. Analysis by capillary GC was performed using a Hewlett/Packard 5880 instrument with splitless injection and a 0.25 mm i.d. 30 m DB-5 (J & W Scientific) column connected to a nitrogen/phosphorous detector. Helium was used as carrier gas for all GC and GC/mass spectrometric analyses at a flow rate of 1 ml/min. Temperature was programmed at 70°C for 4 rain, followed by 5°C/min to 250°C. Recovery studies were conducted by spiking l-g samples of whole trout fry with 1 ~g each of quinoline and 5-hydroxyquinoline. Recovery efficiencies were 90 +__4070 and 51 _+ 6% (n = 4), respectively. Subsequent determinations revealed that recoveries of 5-hydroxyquinoline could be increased to about 80°7o if a second acetate derivatization was conducted after the HCI cleanup step.

225

Mass spectrometric analysis of metabolites Mass spectrometric analysis of acetate derivatives o f products from the hydrolysis o f quinoline metabolites was performed with a Hewlett/Packard 5970B mass selective detector, connected to a H / P 5880 gas chromatograph containing a 60 m x 0.25 mm DB-5 capillary column (J & W Scientific). Chromatographic conditions were essentially the same as those described in the preceding section for the tissue analysis. Mass spectra were acquired at 0.8 sec per scan over the mass range of 40-550 atomic mass units. For determination o f isotope ratios at masses 203, 204, and 205, the mass selective detector was adjusted to the multiple ion monitoring mode. Data were collected for these masses at dwell times of 100 msec. Accurate mass determination of acetates from hydrolyzed metabolite were obtained by high resolution GC/high resolution MS, using a Vacuum Generators ZAB-I F high resolution mass spectrometer interfaced to a H / P 5840A GC, containing a 30 m x 0.25 mm J & W SE-54 capillary column (J & W Scientific). Chromatography was initiated at 50°C and the temperature was programmed at 8°C/min to 280°C. For accurate mass measurements, the instrument was tuned to a static resolving power of 1:10,000. Perfluorokerosene (PFK) was bled into the ion source as a reference mass internal standard. The accurate masses of acetylated metabolites having nominal masses of 203 were obtained by peak matching the signals appearing on an oscilloscope against the PFK known mass of 192.98882, corresponding to C5F7.

Enzyme hydrolysis of metabolite conjugates A sample of one to three gall bladders plus bile from 10-g juvenile trout were extracted three times with 1 ml methanol. The volume was reduced to 1 ml, and a 100-#1 aliquot was hydrolyzed. The hydrolysis procedure generally followed that described by Varanasi et al. (1981). The sample was evaporated under a stream o f nitrogen in a 25-ml centrifuge tube and treated with 2 ml 0.5 N sodium acetate buffer (pH 5) and 2 ml water, and then with 0.5 ml glucuronidase. After mixing, the sample was heated at 37°C for 4 h, then neutralized with sodium carbonate and extracted four times with 2.5-mi portions of ethyl acetate. The combined solvent extracts were evaporated to 100/~1 in preparation for thin layer chromatography. Some o f the enzyme hydrolysis reaction mixtures were analyzed for liberated quinoline metabolite hydrolysis products by adjusting the pH to 12 with 5 N NaOH, derivatizing the mixture with acetic anhydride, and quantifying using gas chromatography as described above.

Thin layer chromatography Quinoline conjugates were chromatographed using two systems: (A) the supernatent layer obtained after shaking butanol/acetic acid/water (40:10:50) was used to develop silica gel 60 F-254 plates (Merck); and (B) the supernatant from butanol/concentrated ammonia/water (40:10:5) was used to develop LK 5 DF

226

4-channel plates (Whatman) with pre-adsorbent strip. System (B) follows Varanasi et al. (1981). In both systems, development chambers were equilibrated for 1 h before the plates were developed or until solvent had migrated 15 cm. Distribution of radiolabeled material on developed plates was determined by removing adsorbent at intervals of 0.5 cm, placing the adsorbent into scintillation vials, and counting.

Statistical analysis Kinetic model theory and non-linear least squares techniques were used to obtain estimates of uptake rates and bioconcentration factors (BCF's). A twocompartment (fish and water) closed system was used as the model for exchange of total radioactivity or parent compound to obtain simultaneous estimates of uptake and elimination from the uptake phase (Hamelink, 1977). RESULTS

Bioaccumulation of quinoline in swimup fry Measurements of quinoline concentration in the exposure water ranged from an average of 0.97 to 1.20 mg/l. Final concentrations after 48 h static exposure were

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115 24

32

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56

64

72

Hoursot Exposure Fig. 1. Uptake and depuration of quinoline in trout ' s w i m u p ' fry (whole body measurements) from I m g / l water column exposure. (A) U p t a k e / d e p u t a t i o n determined by scintillation counting of radiolabel. (B) Percent of radiolabel accounted for as quinoline by direct GC analysis. (C) Measured quinoline concentrations in water column. Values shown as mean _+ standard deviation.

227

92°7o of those initially measured (Fig. IC). Concentrations of ~4C in the exposure water followed a similar trend. Quinoline was not detected in the exposure water during the 24-h depuration period and maximum measured radioactivity during depuration was < 1070 of initial levels. Whole body levels of t4c in juvenile trout increased rapidly during the first 4 h of exposure and reached an apparent plateau after about 24 h (Fig. 1A). Maximum concentrations of quinoline plus metabolites in the whole fish were about nine times those in the water after 36 h of exposure. Rapid elimination of 14C occurred during the first 2 h of the 24-h depuration period (Fig. IA). Whole body concentrations of radioactivity during depuration remained 31 to 38070 of maximum levels measured during the uptake phase. Concentrations of radioactivity accounted for as unmetabolized quinoline by gas chromatography declined steadily during the 72-h test period (Fig. IB). Less than 2% of the radioactivity remained in the form of quinoline after the 24-h depuration period. Concentrations of unmetabolized quinoline in the fish (4.4/~g/g at 16 h exposure) peaked 20 h earlier than the concentration of the radioactive label. Estimated bioconcentration factors (BCF's) of quinoline plus its metabolites (total radioactivity) and of quinoline alone, based on direct analysis of quinoline by GC, were determined on the basis of kinetic model theory. Values closely approximated those obtained by the ratio of measured tissue to water concentrations (Table I). Uptake rate coefficients (Kt) of quinoline were about twice those obtained from the total radiolabel (quinoline plus metabolites). Although elimination rate coefficients (/(2) could be calculated from uptake data for both quinoline and total

TABLE I Estimates of uptake and elimination rate constants and biocentration factors of quinoline plus metabolites (total radioactivity) and parent compound alone. Determinations based on whole body extracts of juvenile rainbow trout, mean weight 0.30 g. All values mean + standard error. Elimination rate constant (k2)

Bioconcentration factor

Compound

Uptake rate constant (k0 a

Based on uptake

Based on uptake

Quinoline + metabolites Quinoline

0.58:1:0.13 h - t

0.073 _+0.022 h - t 0.074_+0.024 h - ~ 7.89 _+ 0.82

7.78 _+ 1.77

1.02_+0.54 h - t

0.27 :i:0.16 h - ~ c

c

Based on elimination b

3.73 _+ 0.43

Based on elimination

" Uptake rate constant estimated from the equation Ca = KI/K2) Cw (1 - e - * 2 , ) where kj is the uptake rate constant, k2 is depuration rate, Cw is the concentration (mg/g) of ~4C or quinoline in the water at steady state, Ca is total radioactivity or parent compound in trout tissue, and t is time. b Elimination rate constant estimated from the equation Ca = Coe-k2,, where Co is total radioactivity or parent compound in trout tissue at time 0. Other values as above. ¢ Elimination rate constant for quinoline was not calculated. All fish collected after I h in clean water contained less than detectable levels of quinoline.

228

radiolabel, an elimination coefficient for quinoline could not be calculated from elimination data, since quinoline in fish sampled at more than 1 h depuration did not contain detectable amounts of quinoline.

Characterization of quinoline metabolites in exposed trout A nitrogen-specific chromatogram obtained during the analysis of the gall bladder and contents from a 10-g juvenile trout exposed to ! mg/l quinoline for 48 h is shown in Fig. 2. In addition to the peak for quinoline, two sets of chromatographic peaks were obtained that were not observed in similar samples from unexposed trout. Mass spectra obtained from compounds producing the first set of three chromatographic peaks (Fig. 2, M + / Z = 187) were indistinguishable from each other, and from the mass spectrum obtained from an O-acetyl-5-hydroxyquinoline standard reference compound, indicating that these compounds were acetates of hydroxyquinolines. Further, the compound corresponding to the first peak in the chromatogram had a GC retention time identical to the acetylated 5-hydroxyquinoline standard compound. The positions of the hydroxyl groups of the other two hydroxy compounds have not been established. However, acetylation experiments with 2-hydroxy-, 4-hydroxy-, and 8-hydroxyquinolines demonstrated that hydroxyl groups on the 2 and 4 positions do not form acetate derivatives under the

M * / Z = 187

M÷/Z = 203 ;,.

• .:

..,.:

,

t-

(/3 r"

C

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;~:

:.; ,,

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Fig. 2. Nitrogen-specific gas chromatogram obtained from analysis of gall bladder and bile from a juvenile trout exposed to 1 mg/l quinoline in water for 48 h.

229 conditions used, and that the 8-hydroxy compound resulted in a derivative with a retention time that did not correspond to any of the fish-derived compounds. Therefore, the two unindentified hydroxyquinolines are substituted in the 3, 6, or 7 positions. The four compounds giving rise to the second set of chromatographic peaks (Fig. 2) were all shown by the mass selective detector to have molecular weights of 203, corresponding to either monoacetylated dihydroxyquinolines or acetylated quinolinethiols. Acetylation was demonstrated from the presence of a base peak in the mass spectra at mass 161, corresponding to the characteristic loss of ketene from the molecular ion: O [C9H6N--S--~--CH3] "+~[C9HTNS] "++ CH2 = C = O The spectra of these compounds obtained from the mass selective detector operating in the repetitive scan mode did not contain the isotope peaks at mass 205 expected for a thiol. However, when the instrument was operated in the multiple ion monitoring mode, monitoring masses 203,204, and 205, the measured ion abundance ratios were close to those predicted for quinolinethiol acetate. For the most prominent component, 204/203 = 12.8 (predicted, 12.58); 205/203 -- 5.0 (predicted, 5.14). Confirmation of identity of the three most abundant S-acetylquinolinethiols was obtained from high resolution mass spectrometry (HRMS). The mass of the molecular ions for those components was 203.0405 amu, corresponding to the elemental composition of the thiol acetate, C~ IH9NOS. The other possible composition, CtIHgNO3, was ruled out from the HRMS data, as it has an exact mass of 203.0582 ainu, well outside (87 ppm) the error for the measurement ( ± 9 ppm). Further confirmation of the presence of sulfur in the compounds having mass 203 was provided in a gas chromatographic experiment. The responses of the three most abundant peaks with M+/Z=203 (Fig. 2) in a sulfur-specific flame photometric detector were observed to have the same retention times as the responses obtained using the nitrogen-specific detector. Only the three most abundant components elicited the sulfur-specific response. The presence of conjugated forms of quinoline metabolites was examined using aliquots of pooled methanol extracts from three gallBbladder/bile samples. Analysis for quinoline metabolite hydrolysis products was performed before and after the sample was hydrolyzed with B-glucuronidase. Fifty-five percent of the radiolabel in an aliquot of the pooled sample could be accounted for as hydroxyquinolines and quinoline thiols using our routine analytical method for tissue analysis (alkaline hydrolysis, acetylation with acetic anhydride, and gas chromatography). The results from thin layer chromatography of another aliquot of the gall bladder extract using procedure (B) are shown in Fig. 3, and are compared with the results from the thin layer separation of the B-glucuronidase hydrolysis products. Before enzyme hydrolysis, 8% of the radiolabel had an Rf in the vicinity of that determined

230

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Fig. 3. Thin layer radiochromatograms obtained, using system B, from untreated and enzymehydrolyzed methanol extracts o f gall bladders from quinoline--exposed juvenile trout. Left, untreated subsamp|e; right, ethyl acetate extract from same size subsample after enzyme hydrolysis with glucuronidase. Migration distances obtained for quinoline derivative standards are indicated by arrows for reference.

for 5-hydroxyquinoline; after enzyme hydrolysis, 23% of the radiolabel was found in the same Rf region. Experiments with 8-hydroxyquinoline glucuronide demonstrated that hydrolysis of the glucuronide with alkali or with enzyme is required for isolation of the hydroxyquinoline acetate using our derivatization procedure. When an aliquot from the pooled gall bladder methanol extract was hydrolyzed with/~-glucuronidase prior to derivatization and gas chromatographic analysis, only two hydroxyacetates were recovered, corresponding to the first two hydroxyquinoline acetate peaks in Fig. 2. No quinolinethiol acetates were recovered after enzyme hydrolysis. The total radiolabel contributed by these two compounds was in agreement with the results from the thin layer experiments. Since the /3-glucuronidase used in the enzyme

231

hydrolysis experiments is known to be contaminated with sulfatase, a gall bladder methanol extract was chromatographed using thin layer procedure (A) in order to determine the most likely conjugated form. Eighty-four percent of the radiolabel had an Rf between 0.06 and 0.18. 8-Hydroxyquinoline glucuronide and 8-hydroxyquinoline sulfate had Rf values of 0.11 and 0.58, respectively and less than 2070 of the label was observed near Rf 0.58. These results indicate that the hydroxyquinoline hydrolysis products are primarily derived from a glucuronide conjugate. Analysis of trout fry used for uptake-depuration experiments using the base digestion/acetylation methodology yielded no measurable quantities of quinoline derivatives. Although sample sizes of whole fish (0.2 to 0.4 g) were small compared to most tissues from the 10-g juvenile trout, the sensitivity of the method should have permitted detection of significant production of quinoline metabolite hydrolysis products.

Distribution o f quinoline and metabolites in juvenile trout Quinoline concentrations in static exposures declined slightly during the 24-h and 48-h uptake periods. Mean concentrations were 1.01, 0.92, and 0.91 mg/I at 0, 24, and 48 h, respectively. Total radioactivity in the exposure water declined from 1864 dpm/ml to 1683 dpm/ml during the same time period. Distribution of quinoline and its metabolites among body parts varied widely from organism to organism. This is reflected in the wide ranges obtained for tissue concentrations presented in Table II. The percentage of total body burden of quinoline and metabolites found in separate body tissues is presented in Fig. 4. Although relative amounts of quinoline and metabolites in individual tissues varied among fish, the observed trends at the three exposure periods were consistent. Body burden of quinoline and its metabolites, as measured by total radioactivity, was greatest after the 24-h exposure period and declined slightly for fish exposed to quinoline for 48 h (Fig. 4). Only a minor portion of the accumulated radioactivity was eliminated after the 24 h depuration. The largest portion of the radioactivity at all time periods was found in the gall bladder. Relative percentage of total radiolabel in muscle tissue declined substantially following depuration. A similar decline in relative body burden was measured for most other tissues (Fig. 4). Total radioactivity over time increased for the carcass, which included remaining tissue (e.g., skin, visceral fat, etc.). Relative concentrations of quinoline and metabolites varied according to tissue (Table II). For example, concentrations of quinoline relative to metabolites in eye tissue remained high (60 to 83°70 of the total radioactivity) throughout the 72-h study period. The majority of the radioactivity in muscle tissue during uptake was parent compound; however, < 1070 was stored as quinoline following 24-h depuration. Concentrations of quinoline in both gill and gut tissue averaged 41 to 47070 of their total radioactivity after 24 and 48 h of exposure and declined to < 1070 and 6070, respectively, of the total following 24-h depuration. In contrast to other tissues,

,.

I

(7-66) 1.4 (1 .o-2.0) 3.4 (2.0-4.4) 0.6 (0.3-1.7) 4.4 (1.0-7.8) 1.0 (0.2-2.2) 6.3 (3.9-9.5)

54

Quinoline


0.4 (0.2-0.7)
ND

70 (41-94) 0.7 (O-l .3) 0.1

OHQuinoline

ND

0.8 (0.3-1.8)
ND

280 (130-480) 1.6 (0.4-2.7) NDb

SHQuinoline 23 (O-69) 13 (8-23) 4.7 (2.8-7.1) 0.5 (O-l .9) 3.7 (O-l 1) 1.4 (0.9-2.2) 2.8 (1.5-5.4)

Unidentified

-

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I__

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’ Sample size (n) ranged from 3 to 6 for each time period. b ND = not detected.

Eye

Gill

Gut

Muscle

Kidney

Liver

Gall bladder and bile

Tissue

24 h’

70 (22-140) 1.4 (0.6-2.4) 0.9 (0.1-2.0) 0.7 (0.2-l .8) 3.2 (0.5-8.0) 0.9 (0.8-1.0) 4.5 (0.6-9.6)

Quinoline

ND

0.5 (O-1.7) ND


360 (130-730) 0.3 (O-0.3) ND

SHQuinoline 110 (o-340) 23 (6-47) 3.6 (2.6-4.6) 0.2 (0.0-0.8) 4.3 (2.0-7.3) 1.0 (0.7-1.6) 0.9 (O-2.8)

Unidentified

- _ - _ ,_ - - - .-

ND

0.3 (O-0.9)

140 (5-290) 0.1 (O-0.3) ND

OHQuinoline

48 h

0.6 (0.2-l .6)

0.1 (O-0.2)
0.3 (O-0.4)
(l-44) ND

17

Quinoline

ND

0.3 (0.1-0.7)

100 (40-180) 0.9 (0.3-I .5) 0.1

OHQuinoline

ND

0.2 (O-0.3)

220 (92-340) 0.8 (O-l .4) ND

SHQuinoline

72 h

230 (9-880) 1.9 (1.3-3.5) 1.1 (0.2-2.6) 0.3 (0.1-0.9) 1.2 (0.5-2.9) 0.4 (0.2-0.8) 0.4 (O-0.9)

Unidentified

Mean and range of concentrations of parent compound and primary metabolites in selected tissues of juvenile rainbow trout. The 24-h and 48-h sample periods represent duration of exposure to 1.O f 0.1 mg/l quinoline in water and the 72-h sample period is after 24-h depuration. Concentrations given as fig/g tissue.

TABLE II

233

55 3 ,ug Equivalent Ouinohne 100

"~

80

.~

~

4 6 1 pg Equivalent Oumohne

Oulnohne

~

Metabolites

~

Unaccounted 14C

~

Rad,ocount,ng

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On=v

g

38 5 h'g Equwalent Oumohne

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O

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~

O

~

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~

-

24-Hr Deputation

Fig. 4. Body burdens of'quinoline and metabolites determined for juvenile trout (n = 4) at three exposure time intervals. To illustrate trends, quinoline, quinoline metabolites, and unidentified metabolites are presented for three individual trout, one at each time period. The total radioactivity recovered (total body burden) is reported as/~g equivalent quinoline, calculated from the specific activity of the quinoline used for the exposure.

< 13% of the radioactivity in liver and bile was determined to be parent compound during the exposure period. Mean concentrations of quinoline and quinoline metabolites 0tg/g) were highest in the gall bladder plus bile (Table II). Relative concentrations of parent compound at 24 h were gall bladder > eye > gut > kidney > liver > gill > muscle and this trend was similar for other time periods. Levels of metabolites were relatively low in all tissues with the exception of the gall bladder plus bile. Sulfur-containing metabolic products were usually found in higher concentrations than hydroxy metabolite hydrolysis products in all tissues. About two-thirds of the identified hydrolysis products in gall bladder plus bile was in the form of the thiol. Concentrations of metabolites increased with respect to quinoline levels throughout the 72-h study (Table II).

DISCUSSION

Our studies showed that juvenile rainbow trout rapidly absorbed and metabolized quinoline from the water column and that uptake and elimination of the parent compound was different than the metabolite fraction. Maximum concentrations of parent compound in fish tissue were reached several hours before maximum concentrations of parent compound plus metabolites (total radioactivity) occurred. Furthermore, while unmetabolized quinoline was rapidly eliminated from the fish after the uptake period, the majority of the metabolite fraction was retained in body tissues throughout the deputation period. The BCF of unmetabolized quinoline

234 estimated from uptake data was about one-half of the BCF calculated from radiolabel data (Table I). However, accumulation of unaltered quinoline was negligible, since quinoline was not detected in fish sampled after elimination periods greater than 1 h. Thus, our results emphasize the importance of conducting elimination experiments coupled with direct analysis of tissues for parent compound when determining the bioabsorption potential of a compound which can be readily metabolized. There have been few reports in the literature of sulfur-conjugated metabolites in fish, although these are the primary transformation products of quinoline found in our studies. Formation of a mercapturate derivative from the herbicide Molinate~ was reported by Lay and Menn (1979). Formation of minor amounts of mercapturic acid derivatives was suggested by thin layer chromatography after interperitoneal injection of coho salmon with naphthalene (Roubal et al., 1977) and after naphtalene feeding (Varanasi et al., 1979). The major metabolite in trout tissues from our experiments is probably a mercapturic acid. Kurachi (1983) found similar thiol derivatives after base hydrolysis of tissues and excreta from rats exposed to PCB's. Subsequent work (Kurachi and Mio, 1983) confirmed that a mercapturic acid, arising from glutathione conjugation, was a primary product. It is unlikely that the sulfur conjugate found in our experiments arises from direct reaction of reduced glutathione with quinoline, or with hydroxyquinoline. Activation of an aryl carbon atom by an electron withdrawing group is considered necessary for reaction, and a hydroxyl group does not sufficiently activate the adjacent carbon atom for glutathione conjugation to occur (Testa and Jenner, 1976). The active intermediate is likely an epoxide, as proposed by Jerina et al. (1968). Foureman and Bend (1984) have identified three closely related species of fish which are particularly proficient at catalyzing the reaction of benzo(a)pyrene 4,5-oxide with glutathione. The epoxide is also a reasonable precursor for the hydroxylated quinolines. However, the structure of the conjugated metabolites may have been altered substantially during the base hydrolysis step used during the analysis. The appearance of a third hydroxyquinoline isomer after base hydrolysis, that is not present after enzyme hydrolysis, suggests that dehydration of dihydrodiols and dihydrothiolols occurs during base digestion of tissue samples. Not all the metabolic products from trout quinoline metabolism were detected using the base hydrolysis/derivatization method. It is unlikely that the unidentified radiolabeled material was completely catabolized, since it was largely extractable from tissues with methanol. Dihydrodiols were a possible metabolite, but they would not have been recovered under our experimental conditions without dehydration to the phenol. Dihydrodiols were found to be major products from the metabolism of naphthalene by starry flounder, Platichthys stellatus (Varanasi et al., 1979). The identity of this unidentified material is of some consequence to human health, since it was a principal constituent of muscle tissue after 24-h depuration, and is therefore available for transfer to humans via the diet.

235 Analysis of specific tissues showed that quinoline and its metabolites were widely distributed throughout rainbow trout body tissues. Absolute amounts of quinoline were highest in muscle tissue during the uptake phase, primarily because the muscle tissue was - 30% of the total body weight. However, levels of quinoline in the muscle did not exceed concentrations in the exposure water during the uptake interval. Concentrations of stored metabolites did not differ appreciably from that of the parent compound during initial stages of exposure. Melancon and Lech (1980) reported that differential elimination of metabolites of 2-methylnaphthalene, but not naphthalene, occurred in muscle tissue of rainbow trout in longer-term studies. The eyes also contained a relatively high concentration of parent compound, possibly due to direct contact with the aqueous medium. Eye tissues of rainbow trout are sensitive to aromatic hydrocarbons. For example, substituted benzenes at similar doses have been reported to manifest certain properties of chemical carcinogenesis in epithelium of the eye lens (Simakhov, 1982). Furthermore, a watersoluble fraction of Wyoming crude oil that contained mainly aliphatic hydrocarbons was shown to induce lenticular and retinal lesions in eyes of cutthroat trout (S. clarkt) (Woodward et al., 1981). The relatively high concentration of parent compound measured in the gut indicated that this tissue may absorb quinoline directly from the water. It is likely that biliary excretion contributed to some of the associated radioactivity, since total water influx via the gut is low in freshwater teleosts. However, since maintenance of ion balance occurs across all permeable surfaces of fish (Bone and Marshall, 1982), uptake of water-soluble materials is not restricted to the gill surfaces. Other studies have reported that the gills are the primary route of uptake of hydrophobic chemicals from the water by fish (Hunn and Allen, 1974; Hamelink and Spacie, 1977; Neff, 1979; Balk et al., 1984). The route of uptake will determine to some degree the form of distribution within the body. For example, compounds taken up by the gills are distributed throughout the circulatory system before passing through the liver, whereas compounds taken up by the gut are routed through the hepatic system after absorption. Biotransformation of chemical forms which can be actively excreted can also influence the extent of accumulation within various tissues (Lech and Bend, 1980). The liver contained relatively low amounts of parent compound, which indicated fairly rapid metabolism of quinoline and excretion of metabolites into the gall bladder. That the bile levels of radioactivity were concentrated 370 to 420 times above exposure water concentrations is also indicative of the importance of hepatic excretion. Greatest concentrations of quinoline plus metabolites were found in the liver and gall bladder after 48-h exposure while highest concentrations in all other tissues occurred at 24 h. Low concentrations of ~4C in kidney are consistent with other studies which indicate renal excretion is a minor route of elimination for organic compounds (Hunn and Allen, 1974). Although freshwater teleosts may pass relatively large volumes of

236

dilute urine (Bone and Marshall, 1982), total water flux when compared to the gills is quite low. However, the kidneys may have a role in excretion of organic compounds that are not reabsorbed in the renal tubules. For example, differential distribution of radiolabel was found in the kidney of northern pike (Esox lucius) exposed to 3H-labeled benzo(a)pyrene and was attributed to the area of urine production (Balk et al., 1984). Kidney excretion may also be limited by the extent of metabolite excretion by the liver into the bile (Klaassen, 1980). High levels of radioactivity associated with the carcass can be partially attributed to the large surface area of the skin and fins which were in direct contact with the water. Thomas and Rice (1982) suggested that high concentrations of ~4C in skin of Dolly Varden (Salvelinus malma) exposed to several aromatic hydrocarbons result from sequestering of metabolites at this site. Similar sequestering of quinoline might account for the steady increase in radioactivity measured in the carcass with time. Disposition and storage in mesenteric fat tissue may also have been a factor in storage and retention of radioactivity in the carcass. The gall bladder was the principle repository for quinoline metabolites at the end of the 48-h uptake period. It is clear from this observation that measurement of bioaccumulation factor from radiocounting alone can introduce two errors. First, there is no discrimination between absorbed compound and its metabolic forms; therefore, bioaccumulation of parent compound is overestimated. Second, storage of metabolic products in the bile is a primary step in their discharge to the environment; thus, making the assumption that this material is bioabsorbed leads to a further overestimate of bioaccumulation. Another consideration is that estimates of bioaccumulation potential are usually limited to the parent compound, but bioabsorbed metabolites are frequently the source of toxicity. The hydroxyquinoline and quinolinethiol hydrolysis products accounted for nearly all the radioactivity in the bile during the two uptake periods and following depuration. Preliminary experiments in our laboratory with tissue fractions of rainbow trout liver have shown that the liver microsomes contain quinoline hydroxylating enzymes similar to those reported in rabbits by Mitoma et al. (1956). Unidentified metabolic products predominated in the liver and kidney, where other enzymes that metabolize foreign compounds are known to occur. It is well established that hepatic microsomal enzymes in fish and higher organisms carry out a number of mixed function oxidase (MFO) reactions that catalyze xenobiotic metabolism (Gillette et al., 1972; Chambers and Yarbrough, 1976; Payne, 1977; Gerhart and Carlson, 1978; and others). Additionally, the activity of these MFO systems can be markedly increased by prior exposure of an organism to many organic chemicals. (James and Bend, 1980; Lech and Bend, 1980). Our studies indicated that the disposition and fate of quinoline and principle metabolites varied both according to time and among tissue types during relatively short-term aqueous exposures. Thus, defining the enzymatic reactions and other factors regulating the fate of these materials under different scenarios in vivo remains an important objective.

237 ACKNOWLEDGEMENTS

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