Pharmacokinetics of 2-naphthol following intrapericardial administration, and formation of 2-naphthyl-β-D-glucoside and 2-naphthyl sulphate in the American lobster, Homarus americanus

xenobiotica , 1997, vol. 27, no. 6, 609 626

Pharmacokinetics of 2-naphthol following

intrapericardial adm inistration, and form ation of

D

2-naphthyl-b- -glucoside and 2-naphthyl sulphate

in the American lobster,

Homarus americanus

C.-L. J. LI and M. O. JAMES*

Department of Medicinal Chemistry, University of Florida, PO Box 100485,

Gainesville, FL 32610, and The Whitney Laboratory, University of Florida,

St Augustine, FL 32086, USA

Received 17 October 1996

1. Following a 0 25-mg kg intrapericardial dose of the phenolic compound, 2-

}

naphthol, to the American lobster, Homarus americanus, a two-compartment model best

"%

described the disposition of parent [ C]-2-naphthol in the haemolymph. Male and female

a

lobsters had similar -phase half lives of 26ꢀ19 min (meanꢀSD, n ¯ 4) and 29ꢀ15 min

b

respectively. The -phase half lives were signi®cantly longer in males, 63 9ꢀ30 9 h, than in

!

females, 30 6ꢀ6 8 h (p 0 05). The total body clearance for females was 26 4ꢀ

!

Õ

"

6 5 ml¬h ¬kg and was higher than that of males, 11 1ꢀ5 9 ml¬h ¬kg (p 0 05).

b d

2. 2-Naphthol was converted to 2-naphthyl- - -glucoside (major metabolite) and 2-

naphthyl sulphate (minor metabolite) such that at 24 h 39 60 6 % of the radioactivity in

b d

haemolymph was 2-naphthyl- - -glucoside, 38 6 58 9% 2-naphthol and 0 5 4 % 2-

naphthyl sulphate.

"

3. The 2-naphthol-derived radioactivity was 99 % bound to haemolymph proteins at

90 % bound at 1 day after the dose, indicating that both 2-naphthol and 2-

"

1 min and

b d

naphthyl- - -glucoside were highly protein bound.

b d

4. 2-Naphthyl- - -glucoside was slowly eliminated from haemolymph in both males

and females, with elimination half lives of 34 78 h. 2-Naphthyl- - -glucoside was the

b d

major metabolite in urine samples collected at 5 days after the dose. Hepatopancreas and

b d

antennal gland contained glucosidase activities, and the long half life of 2-naphthyl- - -

glucoside could be explained by conjugation deconjugation cycling.

!

5. 2-Naphthyl sulphate was eliminated from haemolymph with a half-life

was excreted in urine.

10 h and

Introduction

Marine crustacean species such as the American lobster, Homarus americanus,

are known to accumulate lipophilic organic chemicals to which they are exposed,

primarily into the hepatopancreas (a storage and digestive organ with functional

similarity to the vertebrate liver; Cobb and Philips 1980) and muscle (James 1982,

1989, Little et al. 1986, James et al. 1995, Johansen et al. 1996). Although

hepatopancreatic microsomes from H. americanus contain cytochrome P450 (James

et al. 1982), monooxygenation in vivo of compounds such as benzo[a]pyrene (BaP)

and other aromatic compounds is very slow, and BaP and related compounds are

retained for long periods by H. americanus (Dunn and Fee 1979, James et al. 1995).

The drugs sulphadimethoxine (Barron et al. 1988, Barron and James 1994) and

erythromycin (M. O. James, unpublished data) were also excreted slowly by H.

americanus, though not as slowly as BaP. In contrast, the simple hydroxylated

* Author for correspondence at Gainesville.

’

0049 8254}97 $12 00

1997 Taylor & Francis Ltd

610

C.-L. J. Li and M. O. James

aromatic compound phenol was not retained by H. americanus (James et al. 1991).

Injected phenol was rapidly eliminated from haemolymph, with a b-phase half-life

of 14 min, and was either excreted unchanged across the gills into tankwater or

metabolized to phenyl sulphate, which was excreted in urine (James et al. 1991).

In vitro studies with phenol and two other phenolic substrates, 2-naphthol and

3-hydroxyBaP, showed that all three substrates formed glucose and sulphate

conjugates in the presence of antennal gland or hepatopancreatic subcellular

fractions from H. americanus (Li and James 1993). There were, however, large

diåerences between phenol, 2-naphthol and 3-hydroxyBaP in the Michaelis

constant, K and maximal rate V

for glucose or sulphate conjugation (Li and

m max

James 1993). K diåerences between phenol and 2-naphthol suggested that their

m

major in vivo metabolites would diåer following exposure to low, environmentally

relevant concentrations of these phenolic substrates, with phenol being pre-

dominantly sulphated and 2-naphthol predominantly conjugated with glucose

(assuming similar distributions into the tissues where biotransformation occurs).

The major physicochemical diåerences between phenol and 2-naphthol are that 2-

naphthol has increased molecular size and lipophilicity, since the pK of the phenolic

a

groups are similar, 9 9 for phenol and 9 5 for 2-naphthol (Weast et al. 1987). The log

octanol water partition coeæcients expressed as log P are 2 84 for 2-naphthol and

}

1 46 for phenol (Leo et al. 1971). The objectives of this study were to determine if

the diåerences in biotransformation of 2-naphthol relative to phenol predicted from

in vitro studies actually occurred in vivo and to examine the eåect of increased

lipophilicity on the distribution and excretion of hydroxylated aromatic compounds

and their glucose and sulphate conjugates by H. americanus. The intrapericardial

route of administration was selected so that the amount of 2-naphthol administered

could be easily controlled. Because previous studies have shown that several

xenobiotic-metabolizing enzymes are aåected by the molt cycle in crustacean

species (Singer and Lee 1977, Li and James 1993), most studies were conducted in

animals demonstrated to be in the intermolt stage.

M aterials and m ethods

Chemicals

"

C]-labelled 2-naphthol with speci®c activities of 4 5 and 9 8 mCi mmol ( 99 % radiochemical

purity), 2-naphthol, 2-naphthyl- - -glucoside, N-ethylmaleimide, glucosidase-free aryl sulphatase

"%

[

}

b d

b

(EC 3\1\6\1) type VI from bacterium (Aerobacter aerogenes), and arylsulphatase-free -glucosidase

(EC 3\2\1\21) type II from almond were obtained from Sigma Chemical Co. 2-Naphthyl sulphate

potassium salt with 93 % purity was purchased from Aldrich Chemical Co. XAD-2 minicolumns were

purchased from Alltech Associate Inc. and Sep-Pak^‡Vac 35 cc (10 g) tC18 cartridges (Waters ^‡) were

obtained from Fisher Scienti®c Co. Centrisart^‡centrifuge tubes with 10 000 molecular weight cutoå

membrane were obtained from Vanguard International Inc. All solvents used were hplc grade and

purchased from Fisher.

Animals

Experiments were conducted at the Whitney Laboratory, University of Florida. Male and female H.

americanus, weight 500ꢀ60 g (meanꢀSD), were obtained from a restaurant supplier and held at the

Whitney Laboratory for at least 3 weeks before use. The molt stage of each experimental animal was

determined just before injection and at the time of dissection by examination of the pleiopods as described

previously for the spiny lobster, Panulirus argus (Altman et al. 1994). The treated animals were held

°

singly in tanks with rapidly ¯owing (4 litre min) seawater at 14ꢀ2 C, except that in experiments to

}

determine excretion into tank water, H. americanus was held for 30 min or 1 h after injection in a small

tank containing 2 litres fresh seawater, and thereafter transferred to a ¯ow-through tank for the duration

of the experiment. Three male H. americanus, used to examine the total recovery after 1 day, were held

Pharmacokinetics of 2-naphthol in the lobster

611

singly in a tank with very slowly ¯owing (1 litre h) seawater and the total seawater was collected for

}

chemical analysis. In the total recovery study, the tankwater was kept cool by a chilling unit to maintain

°

the water temperature at 12 5ꢀ1 C. Some of the H. americanus were externally cannulated from their

nephropores to collect urine (Holliday 1977, Snyder and Chang 1991).

Treatment

Five groups of H. americanus, n ¯ 3 or 4, were studied. All animals were injected intrapericardially

l

with 1 ml kg saline solution (0 9 % NaCl) of ["% C]-2-naphthol, 0 25 mg ml containing 7 8 or 17 Ci ml,

}

to give a dose of 0 25 mg 2-naphthol kg. Two groups of male and two groups of female intermolt H.

}

americanus were dosed and sampled at 1 or 5 days after the dose. A group of three pre-molt male H.

americanus were injected with 2-naphthol and sacri®ced 1 day after the dose. Serial haemolymph samples

(1 2 ml) were taken from the appendage joints at diåerent time points. Duplicate 0 1-ml aliquots of each

haemolymph sample were pipetted immediately into vials containing Ecolume^‡scintillation cocktail

(ICN Biomedical, Inc., Costa Mesa, CA, USA), and the remainder of each sample was placed in a tube

containing 0 1 g N-ethylmaleimide to inhibit clotting. Animals were anaesthetized by placing on ice

before sacri®ce and terminal haemolymph samples (3 6 ml) were removed from the pericardial sinus.

Terminal urine samples from uncannulated lobsters were collected from urinary papillae, and other

°

organs were dissected and stored at ®20 C before analyses. Small or discrete organs were weighed

separately, and their actual weights used in percentage dose calculations. For haemolymph, muscle, shell

and shell membrane, the weights used were derived from the mean percentage weights of the body weight

that these tissues represented, i.e. 21 8, 40 5, 22 and 6 % respectively (Barron et al. 1988). The density of

the haemolymph was assumed as 1 g ml. Triplicate 2-ml samples of the 30-min tank water were directly

"%

assayed for [ C].

}

Tissue distribution

Samples of haemolymph and urine (0 1 ml) were directly assayed for ["% C] by liquid scintillation

spectrometry. Duplicate organ samples, 0 1 g, were completely oxidized by the Packard Tricarb^‡tissue

oxidizer to "% CO or digested in 0 5 ml 2 N NaOH and the digest neutralized prior to liquid scintillation

#

spectrometry for quantitation of ["% C]. The percentage dose recovered in each organ or tissue was

calculated from the dpm g in each sample multiplied by the organ weight.

}

Chemical analyses

The haemolymph samples, 1 2 ml, were deproteinized by thoroughly mixing with 2¬3 ml

m

acetonitrile. To the pooled acetonitrile extracts, 5 ml 0 1 ammonium chloride ammonia buåer, pH 10

}

(buåer A), was added and the acetonitrile removed by evaporation under nitrogen. The solution was

made up to 15 ml with buåer A and applied to an XAD-2 minicolumn. The XAD-2 minicolumn was

washed sequentially with 5 ml deionized water, 5 ml methanol, 10 ml deionized water and 10 ml buåer

A before use. After loading the haemolymph extract sample, the column was washed with 15 ml buåer

A then a 10 ml mixture of acetone buåer A, 3 :7, and a 20 ml mixture of chloroform methanol, 3: 1. The

}

last step was to wash the column with 10 ml chloroform methanol, 3 : 1. An aliquot of 1 ml of each eluent

was directly assayed for ["% C] by liquid scintillation spectrometry. The elution pattern of 2-naphthol was

}

"%

determined with [ C]-2-naphthol and the patterns of elution of the unlabelled conjugate standards were

determined by comparing UV scans (400 200 nm) of each fraction with those of the conjugate solutions.

The metabolite composition of each haemolymph extract was further veri®ed by concentrating fractions

for metabolite hydrolysis, as described below.

In some studies, urine samples, 2 4 ml, were made up to 15 ml with buåer A and fractionated by

XAD-2 chromatography as described above for haemolymph. The metabolite composition of each urine

extract was veri®ed as described below (metabolite hydrolysis).

Samples of tank water that held H. americanus for the ®rst 30 min or 1 h, 20 ml each, were extracted

with 2¬10 ml water saturated ethyl acetate. The ethyl acetate extract was evaporated to dryness under

nitrogen, then the residue was dissolved in the mobile phase prior to reversed-phase hplc analysis.

Sep-Pak^‡tC cartridges were used to retain the radioactive materials in the seawater from the total

")

recovery studies. The cartridges were pre-conditioned with 10 vols (350 ml) acetonitrile, then 10 vols

deionized water before use. One peristaltic pump was used as drawing force to facilitate both the loading

and eluting processes. Seawater was assayed for ["% C] before and after the tC cartridge to make sure the

cartridge was not overloaded. After loading the seawater, 100 ml methanol was used to elute the

radioactive materials from the cartridge. The eluents were collected as 10-ml fractions and 0 1 ml was

")

"%

assayed for [ C] by liquid scintillation spectrometry. Fractions with radioactivity were pooled and

evaporated to dryness ; residues were dissolved in the hplc mobile phase before hplc analysis.

The purity of each administered compound or substrate and the identity of each metabolite or

reaction product was checked by hplc (Beckman 110B hplc pump or ISCO 2300 hplc pump) with UV

(Beckman 153 analytical UV detector or Rainin Dynamax model UV-1 UV detector set at 254 nm) and

612

C.-L. J. Li and M. O. James

radiochemical (Radiomatic Flo-one beta radioactivity ¯ow detector) detection. The hplc column was an

Altex 5¬0 46 cm Ultrasphere octadecylsilane guard column coupled to a 25¬0 46 cm analytical

m

Ultrasphere octadecylsilane column. The mobile phases used were 10 m potassium phosphate,

m

pH 2 75 acetonitrile 3 :2, 10 m potassium phosphate, pH 2 75 acetonitrile 7 : 3, and 10 m potassium

phosphate, pH 2 75 acetonitrile 4: 1 (v v) and the ¯ow rate was 1 ml min.

m

}

Metabolite identi®cation and hydrolysis

The metabolite composition of urine, urine extract or haemolymph extract was investigated by hplc

analysis and by determining the stability of the metabolites to several hydrolytic enzymes and to dilute

acid.

m

acetate buåer, pH 5, or 1 Tris HCl, pH 7. The urine solutions were incubated with 0 78 units

Samples of urine or urine extracts were adjusted to the appropriate pH with 0 1 vol. 1

sodium

m

b

-

glucosidase type II from almond (pH 5) or 0 14 units aryl sulphatase type VI from bacterium (Aerobacter

}

°

aerogenes) (pH 7) at 37 C overnight then re-analysed by hplc. Dried urine samples were also incubated

with 2 HCl at 37 C overnight or 6 HCl at 95 C 6 h, neutralised and re-analysed by hplc.

°

m

The activity of each of the hydrolytic enzymes under the above incubation conditions was monitored

m

haemolymph extracts, or buåer and monitoring enzyme-catalysed liberation of p-nitrophenol. After

by adding the appropriate 10 m

p-nitrophenyl conjugates to separate control urine samples,

m

incubation, 0 1 NaOH was added. The amount of p-nitrophenol formed was determined by comparison

of the absorbance at 420 nm with a standard curve of similarly prepared samples of p-nitrophenol at

known concentrations.

Haemolymph protein binding

H. americanus was dosed with 0 25 mg kg [ C]-2-naphthol containing 17 Ci kg and samples of

"%

l

}

haemolymph were taken at 1 min, 1 and 5 days after injection. Duplicate 0 1 ml portions of each sample

were immediately placed into vials containing Ecolume scintillation cocktail. The remaining hae-

molymph, 2 ml, was placed immediately in the lower chamber of a prechilled Centrisart^‡tube containing

a 10 000 molecular weight cutoå membrane attached to the upper tube. The Centrisart tubes were

°

centrifuged immediately at 700 g for 10 min in a refrigerated (4 C) centrifuge. Under these conditions,

3 8 % of the total sample volume was recovered as ultra®ltrate. Samples of the ultra®ltrate collected in

the upper tube were analysed for ["% C] by scintillation spectrometry.

b

-Glucosidase and aryl sulphatase assays

Microsomal and cytosolic subcellular fractions of the hepatopancreas (HP) and antennal gland (AG)

b

-

of control H. americanus were prepared as described in Li and James (1993). A typical assay tube for

m

glucosidase (Lucier and McDaniel 1972) contained 0 05

sodium acetate buåer, pH 4 5 (for HP

microsomes and cytosol) or 0 05 Bis-Tris buåer, pH 6 5 for male AG microsomes and cytosol, pH 6 0

lm b d

m

for female, 0 02 0 1 mg protein, 8 260

2-naphthyl- - -glucoside and deionized water to a ®nal

°

volume of 0 5 ml. The reactions were started by adding the substrate and incubated at 35 C for 30 or

60 min. For aryl sulphatase (Nelson and Daniel 1979), assay tubes contained 0 05 Tris, pH 7 8, 0 15 mg

m

lm

The reactions were initiated by adding the substrate and incubated at 35 C for 45 min. The

HP microsomal protein, 19 150

2-naphthyl sulphate and deionized water to a ®nal volume of 0 5 ml.

°

b

-

glucosidase and sulphatase assay reactions were terminated by addition of 2 ml ice-cold methanol. The

formation of 2-naphthol was measured by ¯uorescence spectrophotometry (excitation at 239 nm,

m

emission at 418 nm) following addition of 0 5 ml 1 NaOH to samples.

Pharmacokinetic analysis

Rstrip^‡software was used initially to estimate parameters that were used to describe the disappearance

of parent compound and the formation of metabolites. A two-compartment model with bolus input, ®rst-

order output and macro-constants as primary parameters from PCNONLIN^‡was used to ®t the parent

compound data.

Statistical analysis

Microsoft Excel^‡software was used to analyse data for statistical signi®cance by Student’s t-test, and

to determine the K and V

max

by linear regression analysis.

m

Pharmacokinetics of 2-naphthol in the lobster

613

Results

Tissue distribution of radioactivity

At 1 and 5 days after the 0 25-mg kg dose, groups of H. americanus were

}

dissected and all tissues analysed for total radioactivity. The results are shown in

table 1 and ®gure 1A and B. Most of the dose remaining in the body at days 1 and

"%

5 was present in the haemolymph. The amounts of [ C] remaining in the

haemolymph and tissues at both 1 and 5 days after the dose were higher in males

!

than females ( p 0 05). In female H. americanus, the percent dose found in the

gonad was very variable between animals (table 1). The percent dose present in the

female gonad correlated directly with the amount of gonadal tissue present in the H.

americanus, regardless of sacri®ce day (®gure 2). Table 1 and ®gure 1A show that 2-

naphthol-derived radioactivity was retained longer by the premolt than the intermolt

males, and that the major diåerence between the premolt and intermolt male H.

americanus was that haemolymph from the premolt animal retained much more 2-

naphthol and metabolites. At both times, and in all groups, the tissues other than

haemolymph with the highest concentrations of radioactivity (ng 2-naphthol

equivalents per g wet weight) were the antennal gland and hepatopancreas (®gures

1A and B). The muscle, the major, edible part of H. americanus, had signi®cantly

lower concentrations of 2-naphthol-derived radioactivity than haemolymph at both

1 and 5 days, in both the male and female. The concentrations of radioactivity per

g intestinal content were not signi®cantly diåerent from those of urine in each

group, although there was considerable inter-animal variability. The concentration

of 2-naphthol-derived radioactivity in the intestinal contents was signi®cantly lower

!

(p 0 05) in the 1 day premolt male than in the 1 day intermolt male or female

lobster (®gure 1A and B).

For most animals, the terminal urine remaining in the urinary bladders at

sacri®ce was collected and found to contain relatively high concentrations of 2-

naphthol and metabolites, but only a small percentage of the administered dose. To

clarify further the role of urinary excretion in elimination of 2-naphthol, attempts

were made to cannulate externally H. americanus to collect all the excreted urine over

a 5-day period. The cannulae of one of these lobsters remained intact for 5 days and

15 5 % of the dose was recovered in the total 5-day urine from the individual female.

"%

In mass balance studies, the excretion of [ C] into seawater was examined in two

premolt and one intermolt males. In these animals 12 4ꢀ3 8 % (meanꢀSD) of the

dose was found in seawater in the 24 h after the dose. A total of 98 8ꢀ4 5 % of the

dose was accounted for in the tissues, intestinal contents, urine and the 1-day total

seawater.

Analysis of Haemolymph

Hplc analyses of the haemolymph acetonitrile extracts showed that 2-naphthol,

2-naphthyl-b-d-glucoside and 2-naphthyl sulphate were present in haemolymph.

The structures of these compounds are shown in ®gure 3. The identities of the

conjugates were con®rmed by enzymatic hydrolyses (data not shown). The

"%

composition of the haemolymph [ C] was routinely quantitated by XAD-2

minicolumn chromatography at each time point sampled from four male and four

female H. americanus over 5 days. The XAD-2 minicolumns retained all of the

glucoside conjugate and 98 % of the parent compound, while the sulphate conjugate

614

C.-L. J. Li and M. O. James

Pharmacokinetics of 2-naphthol in the lobster

615

(a)

(b)

"%

Figure 1. Tissue concentrations of [ C] in male and female H. americanus dosed intrapericardially with

l

0 25 mg kg 2-naphthol labelled with 7 8 or 17 Ci [14C] kg. (A) males ; (B) females. HL,

}

haemolymph ; MUS, muscle ; HP, hepatopancreas ; INT, intestine ; IC, intestinal content; AG,

antennal gland; UR, urine ; GL, gill; GON, gonad. Values shown are meanꢀSE, n ¯ 3 for

lobsters sacri®ced 1 day after the dose, n ¯ 4 for lobsters sacri®ced 5 days after the dose. No urine

samples were collected for 1-day premolt males. One 5-day female was externally cannulated from

the nephropores to collect total urine. All other urine samples are from urine remaining in the

bladders at the time of sacri®ce.

was not retained by the column. The 2-naphthyl-b-d-glucoside was eluted from the

column by mixture of acetone 0 1 m ammonia buåer, pH 10, 3 : 7. 2-Naphthol was

}

eluted by mixture of chloroform methanol, 3 :1. The sulphate conjugate data was

}

corrected for non-retained parent compound.

Analysis of the 1-day haemolymph samples from the intermolt and premolt

males showed that 2-naphthyl-b-d-glucoside was the major metabolite in both

groups (table 2). The haemolymph from premolt males had signi®cantly higher

concentrations of glucoside conjugate than the intermolt animals.

616

C.-L. J. Li and M. O. James

Figure 2. 2-Naphthol-derived ["% C] in the female gonad of the 2-naphthol-treated American lobsters,

H. americanus. Animals were intrapericardially dosed with 0 25 mg kg 2-naphthol and sacri®ced

}

at 1 or 5 days after dose. Individual values are shown. Regression analysis gave a straight line ®t

with r ¯ 0 94.

Figure 3. Pathways of metabolism of 2-naphthol in the American lobster, H. americanus. I, 2-naphthol ;

b

-

b d

II, 2-naphthyl- - -glucoside ; III, 2-naphthyl sulphate. UGT, UDP-glucosyltransferase ; G,

glucosidase ; ST, PAPS-sulphotransferase ; S, sulphatase.

Identi®cation of urinary metabolites

Urine was routinely analysed by direct hplcÐtwo metabolites were found but no

parent compound was detected. The major metabolite found in terminal urine

collected at day 5 co-migrated with 2-naphthyl-b-d-glucoside when analysed by

"%

hplc (table 3), and comprised 90 97 5 % of the [ C] in these terminal urine samples.

Treatment of urine with b-glucosidase did not result in hydrolysis of this peak to 2-

naphthol. Urine extracts obtained by XAD-2 and Alumina N Sep-Pak^‡chroma-

tography resulted in metabolite fractions that were only partially hydrolyzed by b-

glucosidase at pH 5 or 2 m HCl to parent compound. Further puri®cation of the

Sep-Pak-puri®ed samples by hplc and collection of hplc fractions resulted in a

metabolite fraction that was completely hydrolyzed to 2-naphthol by b-glucosidase,

thus con®rming the identity of the glucoside conjugate. The other urinary metabolite

"%

co-migrated with 2-naphthyl sulphate and constituted 2 9 7 % of the excreted [ C].

This minor metabolite was hydrolyzed by aryl sulphatase type VI at pH 7 to the

Pharmacokinetics of 2-naphthol in the lobster

617

Table 2. Composition of the haemolymph of the premolt and intermolt male lobster sacri®ced 1 day

after dose.

Compound

Premolt

Intermolt

2-Naphthyl sulphate

b d

2-Naphthyl- - -glucoside

2-Naphthol

38 1ꢀ36 8

509 6ꢀ62 1

140 5ꢀ61 3

5 2ꢀ4 2

294 8ꢀ39 5

93 1ꢀ17 2

a

Concentrations are in ng ml and all values are meanꢀSD, n ¯ 3.

}

a

b

The premolt lobster had signi®cantly higher concentrations of -naphthyl glucoside than the

!

intermolt animal (p 0 05).

Table 3. Chromatographic separation of 2-naphthol, its conjugates, and other related compounds by

reversed-phase hplc. The hplc systems used were described in the Materials and methods.

Retention time (min)

Compound

M1

M2

M3

1,4-Naphthoquinone

1,2-Naphthoquinone

1,3-Dihydroxynaphthalene

2-Naphthyl sulphate

b d

2-Naphthyl- - -glucoside

2-Naphthol

Major urinary metabolite

30-min or 1 h seawater

24 h seawater

12 4

7 2

7 8

3 3

Ð

5 0

5 2

29 7

5 2

Ð

15 1

18 4

Ð

18 4

Ð

3 4

13 5

3 4

13 5

3 3

Ð

15 1, 18 4

m

M1, mobile phase was 10 m potassium phosphate, pH 2 75 acetonitrile (3 : 2 by volume). M2,

mobile phase was 10 m potassium phosphate, pH 2 75 acetonitrile (7 :3 by volume). M3, mobile phase

}

m

}

m

was 10 m potassium phosphate, pH 2 75 acetonitrile (4 :1 by volume).

}

parent compound. The complete 5-day urine collected from a female H. americanus

"%

contained 84 7 % of the [ C] as 2-naphthyl-b-d-glucoside and 15 3 % as 2-naphthyl

sulphate. There was no evidence for hydroxylated metabolites of 2-naphthol or

conjugates of hydroxylated metabolites in the lobster urine.

Excretion into tank water

Tank water was collected for 30 min or 1 h after the dose for individual lobsters

"%

dosed with 0 25 mg kg [ C]-2-naphthol. This water was analysed for [ C] and

"%

}

chemical composition. For males, 7 7ꢀ5 1 % (meanꢀSD, n ¯ 7) of the dosed

radioactivity was found in the 30-min or 1-h tank water, and 9 0ꢀ5 3 % (n ¯ 7) for

females. There was no diåerence in the amounts found at 30 min or 1 h, so the

results for each sex were pooled. The radiolabelled material excreted into the tank

seawater in the ®rst 30 min was shown by extraction and hplc analysis to consist

primarily of parent compound (table 3).

In the 1-day mass balance studies, 12 4ꢀ3 8 % (n ¯ 3) of the total dosed

radioactivity was recovered in the 24-h seawater. This seawater was passed over tC

")

95 % of the [ C] was retained. Hplc analyses of the

"

"%

Sep-Pak cartridges and

methanol eluates from the cartridges indicated that 55 8ꢀ4 2 % (n ¯ 3) of the

radioactivity in 24-h seawater was 2-naphthyl sulphate and 40 6ꢀ1 9 % was 2-

!

naphthyl-b-d-glucoside (table 3). The chemical nature of the small amount ( 5 %)

of the radioactivity in 24-h seawater that did not bind to the cartridges was not

determined.

618

C.-L. J. Li and M. O. James

Table 4. Pharmacokinetic parameters estimated from the two-compartment model for elimination of 2-

naphthol from haemolymph following an intrapericardial dose.

Parameter

Units

min

Sex MeanꢀSD (n ¯ 4)

a

Distribution phase, t

Elimination phase, t

,

M

F

26ꢀ19

29ꢀ15

/

" #

c

b

,

h

M

F

63 9ꢀ30 9

30 6ꢀ6 89

/

" #

d

a

Õ

"

V

ml¬kg

Õ

M

F

297ꢀ69

342ꢀ94

c

d

b

V

M

F

560ꢀ160

780ꢀ200

p

d

c

Õ

"

Whole body clearance

ml¬h ¬kg

M

F

11 1ꢀ5 9

26 4ꢀ6 5

c

Õ

"

Intercompartmental clearance ml¬h ¬kg

M

F

553ꢀ532

498ꢀ503

a

b

c

d

V is the volume of the central compartment.

V is the volume of the peripheral compartment.

!

Volumes of the central compartment are signi®cantly lower than the volumes of the peripheral

c

p

Male values are signi®cantly lower or slower than female ones, p 0 05.

!

compartment, p 0 05.

Elimination of 2-naphthol from haemolymph

A two-compartment model was used to ®t the data for 2-naphthol concentration

in haemolymph at all times. The parameters estimated from this two-compartment

model are summarized in table 4 and the disappearance of 2-naphthol from

haemolymph is shown in ®gure 4. The half-lives of the distribution phase, a, were

very similar for both males 26ꢀ19 min (n ¯ 4), and females, 29ꢀ15 min, but for the

elimination phase, b, males had longer half lives, 63 9ꢀ30 9 h, than females,

!

30 6ꢀ6 8 h (p 0 05) (table 4). For both male and female, the volume of the central

compartment was signi®cantly lower than the volume of the peripheral compartment

!

Õ

"

( p 0 05). The whole body clearance was lower in males, 11 1ꢀ5 9 ml¬h ¬kg ,

!

Õ

"

than females, 26 4ꢀ6 5 ml h ¬kg (table 4) ( p 0 05).

Elimination of 2-naphthol metabolites from haemolymph

The elimination from the haemolymph of the major metabolite formed in vivo,

2-naphthyl-b-d-glucoside, is shown in ®gure 5. The concentration of 2-naphthyl-b-

d-glucoside in haemolymph peaked between 4 and 8 h after the dose and then slowly

declined. The terminal elimination half lives of 2-naphthyl-b-d-glucoside were

calculated by least-squares analyses of the haemolymph concentrations of 2-

naphthyl-b-d-glucoside from 8 to 120 h and were 53 0ꢀ18 2 h for males and

41 3ꢀ5 3 h for females.

The amount of 2-naphthyl sulphate detected in haemolymph at all time points

was very low, between 0 5 and 4 % of the total radioactivity in haemolymph. In

haemolymph samples from time points after 16 h the amounts of the minor

metabolite, 2-naphthyl sulphate, were close to the detection limit. While it was not

possible to determine the elimination half-lives of 2-naphthyl sulphate from

haemolymph with great accuracy, because of the limited data set, least-squares

analysis of the available data gave terminal elimination half lives in males and

females in the range 7 10 h.

Pharmacokinetics of 2-naphthol in the lobster

619

Figure 4. Elimination of 2-naphthol from the haemolymph of the American lobster, H. americanus.

l

Animals were intrapericardially dosed with 0 25 mg kg 2-naphthol containing 17 or 7 8 Ci

}

[14C] kg. Values shown are meanꢀSD, n ¯ 4. E , female ; D , male.

}

b d

Figure 5. Haemolymph concentrations of 2-naphthyl- - -glucoside in the American lobsters, H.

americanus, following 2-naphthol administration. Animals were intrapericardially dosed with

0 25 mg kg 2-naphthol. Values shown are meanꢀSD, n ¯ 4. The slopes of the lines were

}

estimated by least-squares analysis of mean haemolymph concentrations from 12 to 120 h after

dose. HL, haemolymph ; E , female, D , male.

Binding to haemolymph proteins

For females, measurement of protein binding by ultra®ltration of haemolymph

collected at varying time points after administration of 0 25 mg kg 2-naphthol

}

showed that dosed radioactivity was 99 6ꢀ0 2 % (n ¯ 4) bound after 1 min,

90 4ꢀ5 6 % after 1 day, and 84 6ꢀ14 3 % after 5 days. For males, 99 7ꢀ0 1 % of 2-

naphthol-derived radioactivity was bound after 1 min, 96 4ꢀ2 0 % after 1 day and

94 2ꢀ2 2 % after 5 days. Thus, parent 2-naphthol (1 min data) was 99 % protein

bound. 2-Naphthyl-b-d-glucoside, the major metabolite accounting for 39 60 6 %

"

"%

of the haemolymph [ C] at 1 day and 29 5 56 6 % at 5 days, must also be

93 %

protein bound. The extent of protein binding of the 2-naphthyl sulphate could not

620

C.-L. J. Li and M. O. James

Table 5. Apparent K and V of UDP-glucosyltransferase (UGT) or PAPS-sulphotransferase (ST)

with 2-naphthol as substrate and -glucosidase (G) and aryl sulphatase (S) for 2-naphthyl

m

max

b

glucoside and 2-naphthyl sulphate in both the male and female American lobster.

V

max

Organ

Fraction (activity)

KM

lm

(pmol min mg

}

Sex

pH

(

)

protein)

Hepatopancreas

Microsomes (UGT)

M

F

10 5

4 6

7 8

58 9ꢀ4 8

89 5ꢀ30 3

129ꢀ38

126ꢀ28

174ꢀ47

173ꢀ24

965ꢀ288 (3)

1040ꢀ614 (3)

125 2ꢀ96 2 (6)

158 4ꢀ128 9 (5)

608 6ꢀ300 8 (4)

231 9ꢀ48 4 (4)

199 4ꢀ151 0 (4)

710 7ꢀ177 9 (4)

Microsomes (G)

Cytosol (G)

M

F

a

M

F

a

b

Microsomes (S)

M

F

37 8ꢀ6 1

a

30 7ꢀ1 5

Antennal gland

Microsomes (UGT)

M

F

7 6

6 5

6 1

6 5

6 1

7 5

6 7

15 8

8 5

1050 (pool of 3)

592 (pool of 3)

a

Microsomes (G)

Cytosol (G)

M

F

87 7ꢀ3 0

4 87ꢀ1 32 (3)

2 16ꢀ1 5 (3)

9 20ꢀ2 14 (4)

2 30ꢀ0 50 (3)

a

25 9ꢀ18 3

19 5ꢀ6 8

12 5ꢀ2 5

69 5ꢀ9 9

112ꢀ36 4

a

M

F

a

Cytosol (ST)

M

F

108ꢀ73 (3)

360ꢀ256 (3)

Data for UGT and ST activities were taken from Li and James (1993) with permission. All values

shown are meanꢀSD (n).

!

a

Male values are signi®cantly higher than female ones (p 0 05).

!

Female values are signi®cantly higher than male ones (p 0 05).

b

!

be determined in these studies as

sulphate conjugate at 1 and 5 days.

4 5 % of the haemolymph radioactivity was the

UDP-glucosyltransferase, PAPS-sulphotransferase, b-glucosidase and aryl

sulphatase activities in H. americanus tissues

Previous studies of UDP-glucosyltransferase (UGT) (EC 2\4\1\35) and PAPS-

sulphotransferase (ST) (EC 2\8\2\1) activities in lobster hepatopancreas and

antennal gland with 2-naphthol as substrate under optimal conditions of tem-

°

perature (35 C), concentration of co-substrate (UDP-glucose or PAPS) and pH are

summarized in table 5. 2-Naphthyl-b-d-glucoside could be formed in both

hepatopancreas and antennal gland, whereas 2-naphthyl sulphate could be formed

in antennal gland but not hepatopancreas. Table 5 also shows the results of the

°

present studies of deconjugating activities, measured at 35 C under optimal

conditions for enzyme activity. Both microsomal and cytosolic fractions from the

hepatopancreas and antennal gland were found to have b-glucosidase (G) activities

with 2-naphthol-b-d-glucoside as substrate. As shown in table 5, the male V

of

max

both hepatopancreas and antennal gland cytosolic fractions were signi®cantly higher

than the female ones and the V of the male antennal gland microsomal fractions

max

were also higher than the female ones. The pH optima for the hepatopancreas and

antennal gland b-glucosidases were diåerent (table 5) and this implies that there are

at least two diåerent forms of b-glucosidase in H. americanus. Aryl sulphatase (S)

activity with 2-naphthyl sulphate was found in the hepatopancreas microsomal

fraction but not in microsomal or cytosolic fractions from the antennal gland. 2-

Naphthyl sulphate was a better substrate in female than male H. americanus (lower

°

and higher V ; table 5).Since the in vitro assays were conducted at 35 C and

max

K

m

Pharmacokinetics of 2-naphthol in the lobster

621

°

the H. americanus were housed in water at 12 5 C, studies of the major conjugating

enzyme activity were conducted at the in vivo temperature. When UDP-gluco-

°

syltransferase activity with 2-naphthol as substrate was measured at 12 5 C in the

presence of hepatopancreatic microsomes from one female lobster, V dropped

max

from a mean of 1040 pmol min mg protein (table 5) to 322 pmol min mg protein,

} }

°

whereas K remained the same, 83 1 lm at 12 5 C as compared with a mean of

m

89 5 lm at 35 C (table 5).

°

Discussion

The tissue distribution and pharmacokinetic studies indicated that 2-naphthol

and its major metabolite formed in the lobster, 2-naphthyl-b-d-glucoside, were

highly retained in haemolymph, poorly taken up by muscle and only slowly

excreted. This was in marked contrast with results with the more lipophilic

xenobiotic, benzo[a]pyrene, which was readily taken up by the hepatopancreas and

muscle and very slowly excreted from haemolymph or tissues (James et al. 1995),

and also in contrast to the structurally related but smaller and less lipophilic

xenobiotic, phenol, which was readily excreted across gills as phenol or in urine as

phenyl sulphate (James et al. 1991). The concentration and total amount of 2-

naphthol in the edible H. americanus muscle was low, indicating that uptake from

the haemolymph was ineæcient. In studies with cat®sh, Ictalurus punctatus, the

related compound 1-naphthol was poorly taken up from blood into muscle, such

!

that muscle concentrations of 1-naphthol and metabolites were

10 % of the peak

plasma concentrations of 1-naphthol (Stehly and Plakas 1992). The factors

governing uptake of xenobiotics from haemolymph to muscle are not well

understood, but are of clear importance in the context of transfer of xenobiotics from

the edible portion of food-producing animals to consumers. The present studies

"

showed that 2-naphthol was highly bound ( 99 %) to lobster haemolymph

proteins. Similarly, 1-naphthol was highly bound (92 %) to cat®sh plasma proteins

(Stehly and Plakas 1992). The role of plasma protein binding in the uptake of

xenobiotics from circulatory ¯uids to muscle is unclear, but xenobiotics that are

highly bound to circulating proteins may be less readily transferred to tissues such

as muscle. The circulating proteins may serve as a storage site for the xenobiotic

(Gibaldi 1991).

The distribution studies revealed a diåerence between male and female H.

americanus in the percentage dose that was retained in tissues and haemolymph at 1

and 5 days, and a diåerence between male lobsters at diåerent molt stages at 1 day

after the dose. The male female diåerence may be explained by considering the

diåerences in glucose conjugation and hydrolysis of glucose conjugates between

males and females (see below). The reason for the diåerence between pre-molt and

intermolt H. americanus in retention of 2-naphthol is not known, but may relate to

the general retention of body constituents in haemolymph and internal organs prior

to molting (Cobb and Phillips 1980). Another factor of interest in considering the

disposition of 2-naphthol-derived radioactivity in tissues was that the female gonad

contained radioactivity in proportion to size. Thus the mature gonad, with eggs

ready for fertilization, retained the 2-naphthol-derived radioactivity. The tendency

of lobster eggs to retain xenobiotics has been noted for other compounds, including

benzo[a]pyrene (James et al. 1995) and 9-hydroxybenzo[a]pyrene (Li and James,

unpublished data), and may be of toxicological importance.

A two-compartment pharmacokinetic model adequately described the plasma

622

C.-L. J. Li and M. O. James

data for elimination of 2-naphthol after intrapericardial administration. The half-

lives for the elimination phase were longer for the males, 63 9ꢀ30 9 h, than for the

females, 30 6ꢀ6 8 h (table 4). The volumes of distribution of the central com-

Õ

"

partment were similar in males, 297ꢀ69 ml¬kg , and females, 342ꢀ94 ml¬kg ,

and were smaller than those of the peripheral compartment (males, 560ꢀ160 ml¬

Õ

"

kg ; females, 780ꢀ200 ml¬kg ). The intercompartmental clearances (males,

Õ

"

553ꢀ532 ml¬h ¬kg ; females, 498ꢀ503 ml¬h ¬kg ) indicated that there

was rapid exchange of 2-naphthol between the central and the peripheral

!

compartments. The whole body clearances were signi®cantly smaller (p 0 05) in

Õ

"

males, 11 1ꢀ5 9 ml¬h ¬kg , than females, 26 4ꢀ6 5 ml¬h ¬kg , and were

much slower than the intercompartmental clearances, in accordance with the slow

elimination of 2-naphthol from the lobsters. High binding to haemolymph proteins

was not likely to be an important determinant of the rate of elimination of 2-

naphthol from the haemolymph. The related compound, phenol, was also highly

"

bound ( 99 %) to haemolymph proteins, but was rapidly eliminated from H.

americanus haemolymph with a half-life of 10 14 min (James et al. 1991). In the

cat®sh, I. punctatus, 1-naphthol was readily eliminated from plasma, with a half-life

!

10 h, despite being highly bound to plasma proteins (Stehly and Plakas 1992).

Analysis of haemolymph samples indicated that 2-naphthol was rapidly

metabolized by H. americanus into 2-naphthyl-b-d-glucoside and 2-naphthyl

sulphate conjugates. No 2-naphthol-b-d-glucuronide was detected in haemolymph

or urine. In I. punctatus, 1-naphthol was rapidly metabolized to the glururonide and

sulphate conjugates, which were eliminated in urine and bile (Stehly and Plakas

1992). The salmon, Oncorhynchus kisutch, metabolized naphthalene to several

metabolites including glucuronide, glucoside and sulphate conjugates of hydroxyl-

ated naphthalene metabolites (Malins et al. 1979). In crab, Maia squinado, exposed

to naphthalene, the urine contained glucoside and sulphate conjugates of 1-naphthol

as well as glucoside conjugates of 1,2-dihydroxyl,1,2-dihydronaphthalene (Corner

et al. 1973). In spot shrimp, Pandalus platyceros, exposed to naphthalene, peaks

which co-migrated on hplc with authentic naphthylsulphate, naphthylglucuronide

and naphthylglucoside were found, although it was discussed by Sanborn and

Malins (1980) that the peak identi®ed as napthylglucuronide was present in

naphthalene-spiked seawater, and may not have been produced in the shrimp.

Unlike ®sh, crustaceans are more likely to form glucoside than glucuronide

conjugates of hydroxylated xenobiotics (reviewed in James 1987, 1994). The tissue

distribution data showed that the antennal gland and hepatopancreas were the

organs with the highest concentrations of 2-naphthol-derived radioactivity (®gure

1A and B). It was therefore likely that 2-naphthol was metabolized by the

conjugating enzymes in the hepatopancreas and antennal gland to glucoside and

sulphate conjugates. Previous studies conducted under optimal in vitro conditions

showed that 2-naphthol-UDP-glucosyltransferase activity was present in hepato-

pancreas and antennal gland (Li and James 1993 ; summarized in table 5). In the

present study, it was shown that even at the in vivo temperature of the lobster

°

(12 5 C), hepatopancreas microsomal UDP-glucosyltransferase activity with 2-

naphthol was quite high, with V approximately one-third of the optimal rate

max

measured at 35 C. By comparing K and V

°

of uptake and metabolism and 2-naphthol, it was clear that the more eæcient

for ST and UGT in the major organs

m

max

pathway of metabolism (low K high V ) was UDP-glucosyltransferase in the

m

max

antennal gland and the hepatopancreas (table 5).

Pharmacokinetics of 2-naphthol in the lobster

623

Although in vitro and in vivo studies suggested that glucose conjugation was the

major conjugation pathway, sulphotransferase activity with 2-naphthol was not

negligible in the antennal gland, and analysis of urine and 1-day seawater showed

that between 5 and 10 % of the dose of 2-naphthol was excreted from the lobster as

2-naphthyl sulphate. Only small amounts of 2-naphthyl sulphate, in the range

0 5 4 % of the total radioactivity in haemolymph and close to the limit of detection,

were found in haemolymph samples. This apparent discrepancy between the

amount of 2-naphthyl sulphate excreted and the amount in haemolymph was

probably due in large part to the rapid elimination of the sulphate conjugate in urine,

as indicated by the high amount of 2-naphthyl sulphate in 24-h seawater (table 5)

and the short half-life of elimination of 2-naphthyl sulphate from haemolymph,

!

estimated at

10 h. Sulphation has been viewed as an aid to urinary excretion

(Mulder 1981) and this minor conjugate was formed in the antennal gland which is

located next to the site of elimination, the urinary bladders (Cobb and Phillips 1980).

The half-life of elimination of the major metabolite of 2-naphthol, 2-naphthyl-

b-d-glucoside from haemolymph ranged from 34 to 78 h in individual lobsters and

was in the same range as the elimination half-life of 2-naphthol. In mammals,

xenobiotics that undergo glucuronidation in the liver, excretion in bile, hydrolysis

by intestinal b-glucuronidase and re-absorption of the parent xenobiotic (i.e.

enterohepatic cycling) tend to have a longer half-life in the body and may undergo

more extensive biotransformation before being eliminated than xenobiotics that do

not undergo glucuronidation and enterohepatic cycling (Sipes and Gandol® 1991).

From the observations that 2-naphthol and 2-naphthyl-b-d-glucoside had similar

elimination phase half-lives and slow whole body clearance it is proposed that

enterohepatic cycling or similar conjugation-deconjugation cycles took place in H.

americanus. This is supported by the detection of b-glucosidase activities in the

hepatopancreas and antennal gland of the lobster (table 5). In the hepatopancreas

and antennal gland cytosolic fractions, the males had signi®cantly higher V

than

max

the females and similar K . Thus males may be expected to deconjugate 2-naphthyl-

m

b-d-glucoside more eæciently than females and this may explain the longer elimin-

ation phase half life of 2-naphthol from male haemolymph (63 9ꢀ30 9 h) relative

to females (30 6ꢀ6 9 h). It is not clear from the present studies whether the

conjugation deconjugation cycle occurs solely in the hepatopancreas or also involves

intestinal deconjugation followed by reabsorption. Conjugation deconjugation

futile cycling has been observed in isolated rat hepatocytes (Kaufmann et al. 1991).

It may also be noted that as well as glucosidase activity, hepatopancreas microsomes

contained 2-naphthyl sulphatase activity, thus the total production of the sulphate

conjugate may be regulated in small part by futile cycling via aryl sulphotransferase

and aryl sulphatase.

No single major route of elimination of 2-naphthol or 2-naphthyl-b-d-glucoside

was identi®ed in these studies, but there was evidence that the urine was the major

route of elimination of 2-naphthyl sulphate, whereas urine and faeces were likely

routes of elimination of 2-naphthyl-b-d-glucoside. It was not clear from these

studies how large a role elimination across the gills played in the elimination of 2-

naphthol or metabolites. In studies where tank water was monitored for radio-

activity, 7 7 9 0 % of the dose was recovered in the ®rst 30 60 min in the form of

2-naphthol, suggesting that part of the 2-naphthol dose was excreted across the gills.

Earlier studies showed that the related compound, phenol, was extensively excreted

across the gills in the unconjugated form (James et al. 1991). When slowly-¯owing

624

C.-L. J. Li and M. O. James

tank water in which dosed, uncannulated H. americanus were held was collected for

24 h, however, only 12 4ꢀ3 8 % of the dose was excreted into tank water. The

radioactivity present in the 24-h tank water consisted of glucoside conjugate,

40 6ꢀ1 9 % of the excreted radioactivity, and sulphate conjugate 55 8ꢀ4 2 %, with

no parent 2-naphthol. Because the lobsters were held in slowly ¯owing seawater

tanks in this study, we hypothesize that the excreted 2-naphthol was reabsorbed by

the animals and converted into conjugated metabolites, which were excreted largely

in urine and perhaps partly across the gills. The relatively high concentrations of

radioactivity in terminal urine samples taken at 1 or 5 days suggested that urine was

an important route of excretion, and the complete collection of urine from one

female lobster (15 5 % dose) con®rmed this, but suggested that another route of

elimination, perhaps the faeces, was also important. Relatively high concentrations

of radioactivity were present in intestinal contents, and in intermolt males the

concentration in intestinal contents rose with time (®gure 1). Intestinal contents

were not analysed for composition, due to the small amount of total radioactivity

present in each terminal sample.

As discussed above, the major urinary metabolite of 2-naphthol co-migrated

with 2-naphthyl-b-d-glucoside, but when urine was treated with b-glucosidase (type

II from almond) the glucoside metabolite was resistant to hydrolysis. When isolated

from urine, the metabolite was hydrolysed by b-glucosidase, suggesting that urine

contained inhibitors of b-glucosidase. The urinary inhibitors were separable by hplc

but not by XAD-2 or Alumina N. Sep-Pak chromatography from the major urinary

metabolite. This ®nding illustrates a potential problem associated with relying

solely on results obtained by enzyme hydrolysis of whole urine for identi®cation of

conjugates. Field et al. (1991) showed that compounds containing imidazole rings

inhibitors of b-glucosidase type II from almonds. It was possible that the lobster

urine contained compounds containing imidazole rings, such as l-histidine, which

had a K ¯ 7 3 lm for b-glucosidase.

i

In summary, 2-naphthol was rapidly metabolized by two competing conjugating

enzymes, aryl sulphotransferase (low capacity) and UDP-glucosyltransferase (high

capacity) and slowly eliminated from the American lobster, H. americanus. Lobsters

that were in the pre-molt stage retained 2-naphthol and metabolites for longer than

those in the intermolt stage, and males retained 2-naphthol and metabolites for

longer than females. Only a small percentage of the dose was taken up into the

muscle. The minor metabolite, 2-naphthyl sulphate, was formed in the antennal

gland, and eliminated from the body in urine. The in vivo and in vitro data suggested

that conjugation-deconjugation cycling occurred for the major metabolite, 2-

naphthyl-b-d-glucoside, which was formed in the hepatopancreas and antennal

gland, and slowly eliminated in urine and faeces and perhaps across the gills. Since

the major diåerences between the phenol (James et al. 1991) and 2-naphthol were

lipophilicity and molecular size, these studies show that lipophilicity and molecular

size play important roles in determining how phenolic compounds are metabolized

by H. americanus in vivo, both quantitatively and qualitatively.

Acknow ledgem ents

We are grateful to Sean Boyle and Allen Altman for their assistance with animal

handling and hemolymph sampling. Special thanks go to Drs M. G. Barron and D.

Weiner for their assistance with the pharmacokinetic modeling and E. Chang for his

Pharmacokinetics of 2-naphthol in the lobster

625

help in demonstrating the process for cannulating the lobsters. This work was

supported by the US Public Health Service, grant ES-05781, and was taken in part

from the PhD dissertation of C.-L. J.L. submitted to the Graduate School of the

University of Florida in partial ful®llment of the requirements of the PhD 1993.

References

ltman

uono

ames

, M. O., 1994, The eåect of ecdysis on DNA of the

hepatopancreas and green gland of the Florida spiny lobster, Panulirus argus. Comparative

A

, A. H., B

, R. J. and J

107B

ames

Biochemistry and Physiology,

arron edutis

, 419 426.

, M. O., 1988, Pharmacokinetic of sulphadimethoxine in the

B

C

, M. G., G

, C. and J

18

lobster, Homarus americanus, following intrapericardial adminstration. Xenobiotica , , 269 276.

arron ames

, M. G. and J

, M. O., 1994, Oral bioavailability of single and multiple doses of

24

hillips

, B. F., 1980, The Biology and Management of Lobsters, vol. I (New York :

sulfadimethoxine to the lobster, Homarus americanus. Xenobiotica , , 921 932.

obb

, J. S. and P

Academic).

orner

, E. D. S., K

ilvington

ara

, C. C. and O’H , S. C. M., 1973, Qualitative studies on the

metabolism of naphthalene in Maia squinado (Ernst). Journal of the Marine Biological Association,

53

U.K., , 819 832

unn

and ee

D

, B. P.

Fisheries Research Board of Canada, , 1469 1476.

aines hrystal uszniak

F

, J., 1979, Polycyclic aromatic hydrocarbon carcinogens in commercial seafoods. J.

35

, E. J. T. and L

imidazoles as glycosidase inhibitors. Biochemical Journal,

ibaldi

ield

F

, R. A., H

, A. H., C

, M. C., 1991, Histidines, histamines and

274

, 885 889.

, M., 1991, Biopharmaceutics and Clinical Pharmacokinetics, 4th edn (Philadelphia : Lea Febiger)

olliday

G

H

, C. W., 1977, A new method for measuring the urinary rate of a brachyuran crab. Comparative

58A

Biochemistry and Physiology,

, M. O., 1982, Disposition and taurine conjugation of 2,4-D,2,4,5-T,DDA and phenylacetic acid

, 119 120.

ames

J

10

in the spiny lobster, Panulirus argus. Drug Metabolism and Disposition, , 516 552.

, M. O., 1987, Conjugation of organic pollutants in aquatic species. Environmental Health

71

Perspectives , , 97 103.

, M. O., 1989, Biotransformation and disposition of PAH in aquatic invertebrates. In Metabolism

Polycyclic Hydrocarbons in the Aquatic Environment, edited by U. Varanasi (Boca Raton: CRC

Press), pp. 69 91.

ames

J

, M. O. 1994, Pesticide metabolism in aquatic organisms. In Pesticides in Ground and Surface

$

Water, edited by H. Borner. Chemistry of Plant Protection, vol. 9 (Berlin: Springer), pp. 153 189.

ltman

i

, A. H., L , C.-L. J. and S

chell r

, J J. D., 1995, Biotransformation, hepato-

pancreas DNA binding and pharmacokinetics of benzo[a]pyrene after oral and parental

, M. O., A

95

administration to the American lobster, Homarus americanus. Chemico. Biological Interactions,

141 160.

,

ames

ritchard

, J. B., 1991, Pesticide metabolism in aquatic species. In Pesticide Chemistry,

Advances in International Research, Development and Legislation, edited by H. Frehse (Weinheim :

VCH), pp. 227 286.

J

, M. O. and P

chell

r

arron i

, M. G. and L , C.-L. J., 1991, Rapid, dose-dependent

, M. O., S

elimination of phenol across the gills, and slow elimination of phenyl sulfate in the urine of phenol-

19

, J J. D., B

dosed lobsters, Homarus americanus. Drug Metabolism and Disposition, , 536 542.

herman isher end

, J. R., 1982, Benzo[a]pyrene metabolism in

, M. O., S

, B., F

, S. A. and B

reconstituted monooxygenase systems contained cytochrome P-450 from lobster Homarus

americanus hepatopancreas fraction and NADPH cytochrome P-450 reductase from pig liver.

22

Bulletin of the Mount Desert Island Biological Laboratory, , 37 39.

ohansen

lexander

ossland

lanting

øvik

aarder

dynia

,

J

, H. R., A

jerve

, J., R

, O. J., P

echer

, G., 1996, PCDDs, PCDFs, and PCBs in human blood in relation

, S., L

, M., G

, P. I., G

W., B

, K. S. and B

to consumption of crabs from a contaminated fjord area in Norway. Environmental Health

104

Perspective ,

auffman

, 756 764.

hittaker

nundi

K

, F. C., W

conjugate by isolated hepatocytes. Molecular Pharmacology, , 414 420.

ansch lkins

, M., A

, I. and Thurman, R. G., 1991, Futile cycling of a sulfate

39

71

, D., 1971, Partition coeæcients and their uses. Chemical Reviews, ,

eo

L

, A., H

525 616.

L , C.-L. J. and J

, C. and E

i

ames

, M. O., 1993, Glucose and sulfate conjugations of phenol, 2-naphtholand 3-

26

hydroxybenzo[a]pyrene by the American lobster, Homarus americanus. Aquatic Toxicology,

57 72.

,

ittle

ames

oureman

hexadecane disposition in the spiny lobster, Panulirus argus and the American lobster, Homarus

eatherby end

, R. P. and B

"%

, J. R., 1986, 1- C-n-

L

, P. J., J

, M. O., F

, G. L., W

6

americanus. Journal of Environmental Pathology, Toxicology and Oncology, , 13 29.

626

Pharmacokinetics of 2-naphthol in the lobster

ucier

c

aniel b

, O. S., 1972, Alterations in rat microsomal and lysosomal -glucuronidase

L

, G. M. and M D

by compounds which induce hepatic drug-metabolizing enzymes. Biochemica et Biophysica Acta,

261

, 168 176.

ollier

hydrocarbons in aquatic organisms : analysis of metabolites by thin layer chromatography and

alins

homas

oubal

, L. C. and R , W. T., 1979, Metabolic fate of aromatic

M

, D. C., C

, T. K., T

high-pressure liquid chromatography. International Journal of Environmental Analytical Chem-

6

istry, , 55 66.

ulder

M

N

, G. J., 1981, Sulfation of Drugs and Related Compounds (Boca Raton : CRC Press).

35

,

elson

aniel

, W. L., 1979, Interstrain variation of murine arylsulfatase C. Experientia ,

, K. and D

309 310.

anborn

alins

, D. C., 1980, The disposition of aromatic hydrocarbons in adult spot

S

, H. R. and M

shrimp, Pandalus platyceros and the formation of metabolites of naphthalene in adult and larval

10

spot shrimp. Xenobiotica , , 193 200.

inger

ee

, S. and L , R. F., 1977, Mixed function oxidase activity in the blue crab, Callinectes sapidus:

tissue distribution and correlation with changes during molting and development. Biological

153

Bulletin,

andolfi

, I. G. and G

, 377 385.

ipes

, A. J., 1991, Biotransformation of toxicant. In Casarett and Doull’s Toxicology,

laassen

edited by M. O. Amdur, J. Doull and C. D. K

88 126.

, 4th edn (New York : Pergamon), pp.

nyder

hang

$

S

, M. J. and C

lobsters, Homarus americanus. Biological Bulletin,

tehly

, E. S., 1991, Metabolism and excretion of injected [ H]-ecdysone by female

180

, 475 484.

, G. R. and Plakas, S. M., 1992, Disposition of 1-naphthol in the channel cat®sh, Ictalurus

20

S

punctatus. Drug Metabolism and Disposition, , 70 73.

stle and eyer

, W. H., 1987, CRC Handbook of Chemistry and Physics (Boca

east

W

, R. C., A

, M. J.

B

Raton : CRC Press), pp. D163.

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Pharmacokinetics of 2-naphthol following intrapericardial administration, and formation of 2-naphthyl-β-D-glucoside and 2-naphthyl sulphate in the American lobster, Homarus americanus

DOI: 10.1080/004982597240389

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