Introduction

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Topically applied compounds may undergo substantial cutaneous metabolism during percutaneous absorption (Noonan and Wester, 1989; Bickers and Mukhtar, 1990; Hotchkiss, 1992). Thus, a compound that penetrates skin may enter the systemic circulation as a metabolite, rather than the parent compound.

Fluroxypyr methylheptyl ester [1-methylheptyl-4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid (FPMH)]³ is marketed as Starane (Dow AgroSciences, Indianapolis, IN). FPMH is manufactured from the intermediate fluroxypyr methyl ester (FPM) and is rapidly hydrolyzed within plants to form fluroxypyr (FP). The metabolism of FP, FPM, and FPMH has been studied during percutaneous absorption through human and rat skin in vitro (Hewitt et al., 2000). Both FPM and FPMH were hydrolyzed during penetration, with only the acid metabolite, FP, being identified in the perfusate. Within the skin itself, the degree of FP formation was directly related to the extent of reservoir formation in the stratum corneum, where only parent ester was recovered. When the esters passed into the viable tissue beneath, hydrolysis was very rapid. FP does not undergo additional metabolism, either in vitro (Hewitt et al., 2000) or in vivo (Dow AgroSciences, unpublished data).

The use of skin homogenates for metabolism studies enables a greater control over metabolic conditions, as well as the use of inhibitors that would be lethal to animals. The latter allows for the identification of the esterases involved in the hydrolysis of these compounds. Because other structurally similar compounds have been shown to be metabolized by carboxylesterases, we hypothesized that FPM and FPMH also would be metabolized by these enzymes. Therefore, two inhibitors of these enzymes were used: a nonspecific inhibitor, bis-p-nitrophenyl phosphate (BNPP) and a specific inhibitor of arylesterases, mercuric chloride. The effect of eserine (physostigmine; a specific inhibitor of cholinesterase) was also compared.

This article describes the metabolism of FP, FPM, and FPMH in vitro, using rat skin homogenates. The maximum rate of hydrolysis of these compounds (V_max) and the Michaelis constant (K_m) were determined. In addition, the esterase(s) involved in the hydrolysis were identified.

	Materials and Methods

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Compounds. [ring-¹⁴C]-FP (specific activity 24 mCi/mmol; radiochemical purity >99%), [ring-¹⁴C]-FPM (specific activity 26.6 mCi/mmol; radiochemical purity >99%), and [ring-¹⁴C]-FPMH (specific activity 25.1 mCi/mmol; radiochemical purity >99%) were supplied by Dow AgroSciences Europe (Wantage, UK). Other chemicals were obtained from standard suppliers of laboratory chemicals.

Skin Preparation. Male Fischer 344 rats (200-250 g) were obtained from Harlan Olac (Oxford, UK), and were maintained on a diet of Biosure (Manea, UK), Labsure CRM (Chow for Rat and Mouse) pellets, (Special Diet Services, Witham, Essex, UK) and water ad libitum. The rats were lightly anesthetized with halothane before sacrificing by cervical dislocation. The dorsal region of the rat was clipped and the area removed. The skin was finely minced and approximately 200 mg (wet weight) was placed into 20-ml plastic scintillation vials containing ice-cold 4.3-ml phosphate buffer (0.1 M; pH = 7.4) and homogenized (three 10-s bursts) using an Ultra-Turrax (T-25) homogenizer (IKA-Werke GmbH, Staufen, Germany). The resulting skin homogenates were kept on ice until required. The whole of this skin homogenate was then transferred to a glass reaction tube, 0.6 ml of NADPH "generator solution" was added (0.42 g of glucose 6-phosphate + 0.112 g of NADP + 2.5 U/ml glucose 6-phosphate dehydrogenase, dissolved in 30 ml of 50 mM magnesium chloride in Tris-HCl, pH = 7.4) and allowed to equilibrate for 5 min at 37°C. Unlabeled test compound (100 µl; FP, FPM, or FPMH dissolved in dimethyl sulfoxide) was added to the reaction tubes at time zero, vigorously mixed, and incubated for different times. At the end of the desired incubation time, 5 ml of 100% acetonitrile (containing 5% 5 N sulfuric acid, which stabilizes any parent ester present within the incubation mixture) was added to stop the reaction and to enable the extraction of metabolites. The extract was centrifuged at 2200g for 20 min. The resulting supernatant was passed through a 13-mm 0.45-µm syringe filter (Whatman, Clifton, NJ) and injected directly onto an HPLC column. The HPLC method was as that described by Hewitt et al. (2000).

Extraction Efficiency. The efficiency of the acetonitrile/sulfuric acid extraction procedure was calculated using radiolabeled FPMH, in a pilot skin homogenate study. A concentration of 7.3 × 10⁴ M FPMH, spiked with 0.5 µCi [¹⁴C]-FPMH, was added to three rat skin homogenates and incubated at 37°C for 24 h. After this time, the mixture was extracted with acetonitrile and the radioactivity in resultant supernatants (10-µl aliquots) were measured by liquid scintillation spectrometry. The acetonitrile extraction efficiency of FPMH, using a radiolabeled standard, was 98.6 ± 5.5% (n = 3).

Metabolism Studies. Three different concentrations of FP (final concentrations = 7.3 × 10⁵, 3.6 × 10⁴, and 7.3 × 10⁴ M), FPM (final concentrations = 9.9 × 10⁵, 5.0 × 10⁵, and 9.9 × 10⁴ M), and FPMH (final concentrations = 7.3 × 10⁵, 3.6 × 10⁴, and 7.3 × 10⁴ M) were added to rat skin homogenates and incubated for 0 min (control 1), 30 min, and 1, 3, 6, and 24 h. The reaction was terminated by the addition of acetonitrile, and the supernatant was analyzed by HPLC for the parent ester and metabolites. This was repeated on three or four separate occasions, using skin from three or four different animals. Four separate negative controls were run concurrently: Control 1. Skin homogenate + test compound at 0-h time point, to assess the efficiency of acetonitrile/sulfuric acid in stopping the reaction and measure any subsequent hydrolysis. Control 2. Test compounds incubated for 24 h in the absence of skin homogenate (i.e., phosphate buffer alone), to measure the extent of nonenzymatic aqueous hydrolysis. Control 3. Skin homogenate samples were boiled for 20 min, to denature the skin esterases. The esters were incubated for 24 h to determine whether the hydrolytic process is an active process, requiring the intact enzymes. Control 4. Blanks were also run for 24 h with skin homogenate present, with 100 µl of dimethyl sulfoxide alone.

Enzyme Kinetic Studies. The kinetics of hydrolysis of FPM and FPMH were followed by measuring the production of FP. FPM (final concentrations = 5.0 × 10⁶ - 9.9 × 10³ M), and FPMH (final concentrations = 7.3 × 10⁶ - 7.3 × 10³ M) were incubated for 0, 2, 5, 10, 20, 30, and 45 min, and 1 and 2 h. This was repeated on two separate occasions using skin from two different rats. Controls (as described above) were incubated in parallel. The Lineweaver-Burke, Eadie-Hofstee, and Hanes-Woolf transformation plots, as well as the computer model, Mac Curve Fit, were used to calculate K_m and V_max. An average of the four methods is presented.

Enzyme Inhibition Studies. Homogenates were preincubated for 10 min with either BNPP, eserine, or mercuric chloride (final concentrations = 0.05, 0.1, and 0.5 mM). FPM (5.0 × 10⁴ M) or FPMH (3.6 × 10⁴ M) was then added and incubated for 20 min. Termination of the reaction and extraction of the metabolites were carried as described above. Two negative controls were included: 1) incubations without rat skin homogenate, and 2) incubations whereby acetonitrile/sulfuric acid was added before the addition of the test compound. Positive controls, containing no inhibitors, were also included for comparison.

	Results

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Metabolism in Rat Skin Homogenates In Vitro.

FP Incubation of FP with rat skin homogenates (at all concentrations) produced no detectable metabolites. In all cases, including controls, there was 100% recovery of FP (data not shown).

FPM. The metabolism of FPM by rat skin homogenates was very rapid and essentially complete after 1 h, at all concentrations tested (Fig. 1A). In contrast, FPM hydrolysis at time 0 h (control 1) was very low at all concentrations (2.7-6.0%). There was 3.3 to 11.3% hydrolysis when no skin was added (control 2); however, the rate of hydrolysis in boiled skin (control 3) was 9.7 to 20.7% and was significantly greater than other control values (P < .05). The HPLC traces from incubations containing no test compound showed no extra peaks that eluted at the same retention times as FP or FPM (control 4). The average total percentage of recovered material (both parent compound and metabolite) from all experiments was 98%. In all incubations, at all concentrations, there were no additional metabolites of FP detectable at any time point.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Metabolism of FPM (A) [9.9 × 104 M (), 5.0 × 104 M (), and 9.9 × 105 M ()] and FPMH (B) [7.3 × 104 M () 3.6 × 104 M (), and 7.3 × 105 M ()] by rat skin homogenates in vitro. Mean ± S.D., n = 4.

FPMH. The metabolism of FPMH by rat skin homogenates was less extensive and less rapid than FPM, at all three concentrations (Fig. 1B). The initial rate of metabolism was rapid but tailed off and became saturated, especially at the highest concentration (7.3 × 10⁴ M). Only 69.4 ± 9.9% of the highest concentration was hydrolyzed in 24 h, compared with approximately 100% hydrolysis of FPM after only 1 h. Parent ester incubated at lower concentrations was also detectable up to 24 h, but this was less than 3% of the initial amount. Approximately 2 to 3% of FPMH (all three concentrations) was hydrolyzed to FP at time 0 (control 1). Between 0.8 and 8.9% of FMPH was hydrolyzed in the absence of skin homogenate (control 2). The rate of hydrolysis in boiled skin (control 3) was 1.2 to 12.6% and was statistically significantly greater than other control values (P < .05). The HPLC traces from incubations containing no test compound showed no extra peaks that eluted at the same retention times as FP or FPMH (control 4). The total percentage of recovered material was 96%. In all incubations, at all concentrations, there were no additional metabolites of FP detectable at any time.

Determination of K_m and V_max. The kinetic parameters, K_m and V_max, were calculated from the linear part of the hydrolysis rate curves (within the first 10 min for both esters) and were expressed as micromoles of FP formed per minute per gram of wet tissue.

FPM. The rates of metabolism of a range of FPM concentrations in rat skin homogenates are shown in Fig. 2A. The linear section was within the first 5 min. From this, the V_max was calculated to be 1398 µmol FP/min/g of wet weight, and the apparent K_m was 251 µM.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Rate of FPM (A) and FPMH (B) hydrolysis by rat skin homogenates at varying substrate concentrations. Mean ± S.D., n = 4.

FPMH. The rates of metabolism of a range of FPMH concentrations in rat skin homogenates are shown in Fig. 2B. The V_max for FPMH hydrolysis was significantly lower (P < .05) than for FPM, reaching an average of 472 µmol FP/min/g of wet weight. The apparent K_m value was 256 µM, and was not significantly different from the apparent K_m observed for the enzyme involved in the metabolism of FPM.

Identification of Specific Esterases.

FPM Preincubation with the carboxylesterase substrate BNPP resulted in a significant decrease in the hydrolysis of 5.0 × 10⁴ M FPM (Fig. 3). This inhibition increased with increasing concentrations of BNPP. The metabolism of FPM was unaffected by preincubation with the cholinesterase inhibitor, eserine, and the arylesterase inhibitor, mercuric chloride. Control incubations in HEPES-buffered Hank's balanced salt solution showed little or no aqueous hydrolysis to FP (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of enzyme inhibitors on the metabolism of FPM (5.0 × 104 M) and FPMH (3.6 × 104 M) by rat skin homogenates (incubated for 20 min). Mean ± S.D., n = 4.

FPMH. As for FPM, preincubation with BNPP resulted in a significant decrease in the hydrolysis of 3.6 × 10⁴ M FPMH (Fig. 3). This inhibition increased with increasing concentrations of BNPP. Metabolism of FPMH was unaffected by preincubation with eserine or mercuric chloride, whereby 50 to 60% of this compound was still hydrolyzed. Control incubations in HEPES-buffered Hank's balanced salt solution showed little or no hydrolysis to FP (data not shown).

	Discussion

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These data show that both FPM and FPMH were extensively metabolized in rat skin homogenates to the acid metabolite, FP. In no instance were any other metabolites detected. FPM was essentially hydrolyzed completely within 1 h (over the concentration range studied), with hydrolysis occurring more rapidly at lower substrate concentrations. FPMH was not as efficiently metabolized as FPM, whereby parent ester was always present after 24 h. These data concur with observations made in whole skin, where the hydrolysis of both FPM and FPMH was very rapid, but only after diffusion from the stratum corneum reservoir (where no metabolism of these esters occurs) into the esterase-rich layers beneath (Hewitt et al., 2000).

The kinetics of the hydrolysis of the two FP esters differed. The V_max was approximately 3-fold higher for FPM than FPMH. The affinity of the esterase enzymes responsible for the metabolism of FPM and FPMH, as indicated by their K_m values, were very similar, 250 and 256 µM, respectively. These K_m values are relatively high (indicating a relatively low affinity), in comparison with other xenobiotic-metabolizing enzymes, for example, S-warfarin 7-hydroxylation by CYP2C9 (K_m 4 µM) and dextromethorphan O-demethylation by CYP2D6 (K_m 5-20 µM) (Rodrigues, 1994).

The cutaneous metabolism of FPM and FPMH in rat skin homogenates was predominantly mediated by an active enzyme system. There was a basal level (1-10%) of nonenzymatic ester hydrolysis in buffer when no skin was present. Hydrolysis in boiled skin homogenates was higher than in buffer incubations, accounting for 10 to 20% and 1 to 13% of the hydrolysis of FPM and FPMH, respectively. This higher rate of hydrolysis may be due to incomplete inactivation of the esterases during boiling, suggesting the robust nature of these enzymes. This may also indicate that the presence of protein (whether functional or not) may catalyze the breakdown of the esters. However, the contribution of this would be considered negligible in comparison with the extensive hydrolysis that occurs within minutes with unboiled homogenates.

The enzymes involved in the metabolism of these two esters were characterized using selective enzyme inhibitors. BNPP is a known substrate for carboxylesterase isoenzymes in rat liver (Mentlein et al., 1988). These data indicate that the affinity of BNPP for the carboxylesterase enzyme involved was far greater than for the two esters. When approximately equal concentrations of each were incubated together (500 µM BNPP compared with 498 µM FPM and 364 µM FPMH), the metabolism of FPM and FPMH was inhibited to 92 and 99%, respectively. This concurs with the calculated K_m values, which showed that both esters have relatively low affinities for the enzyme involved in their metabolism. Eserine, an anticholinesterase, had no inhibitory effect on the hydrolysis of FPM and FPMH. Mercuric chloride, an inhibitor of enzymes that contain a sulfhydryl group (cysteine) at the active site, such as A esterases and arylesterases, also had no effect on FPM and FPMH hydrolysis. Thus, the enzyme involved in the metabolism of FPM and FPMH is a carboxylesterase.

A study by McCracken et al. (1992) established that carboxylesterases are located in the cytosolic fraction of rat skin and are easily released by homogenization. In their investigations, A-esterases (except for arylesterase activity) played no role in ester hydrolysis, and the involvement of cholinesterases was minimal. Other studies have found significant levels of carboxylesterase in rat skin (and human skin). For example, the metabolism of the pesticides fluazifop-butyl, carbaryl, and paraoxon by the rat was reported to be via carboxylesterases within many different tissues, including skin (McCracken et al., 1993).

It is also feasible that other enzyme systems may have been involved in the hydrolysis of these two esters, such as monooxygenases, alcohol dehydrogenases, and glutathione S-transferases (Price, 1991), which have all been shown to have a certain degree of carboxylesterase activity. Albumin has also been reported to catalyze the hydrolysis of certain esters, namely para-nitrophenyl esters and aryloxy propionic esters (Kokubo et al., 1982; Kamal et al., 1991). Additional inhibitory studies are needed to determine the role of other routes of hydrolysis in the metabolism of FPM and FPMH. Based on previous percutaneous absorption studies (Täuber and Rost, 1987; Clark, 1993; Hewitt et al., 2000), it is likely that FPM and FPMH will be extensively metabolized by human skin, although to a much lower extent.

In conclusion, the toxicological ramifications of these data presenting the cutaneous metabolism of the herbicides FPM and FPMH will be of great importance because the major route of human systemic exposure to these types of compounds will be via the skin. Taken together with data presented by Hewitt et al. (2000), differences in the rate of metabolism of these two esters may influence the rate and extent of their penetration through rat and human skin. The partition coefficient between the stratum corneum reservoir and the viable epidermis may be increased if the compound is removed from the viable tissue faster (thereby increasing the concentration gradient). The rate of compound removal is dependant on its rate of metabolism, thus linking metabolism to penetration. This was reflected in the higher percentage of FPM absorption into the viable skin than the more slowly metabolized FPMH (Hewitt et al., 2000). The metabolism studies presented in this article show that the skin is capable of rapid and complete hydrolysis of these two esters. No parent ester passed through the viable layers of the skin (Hewitt et al., 2000), only the acid metabolite, FP. Therefore first pass metabolism will be complete before these compounds reach the systemic circulation.