Conjugated-PCP

PCP clearance was essentially metabolic, with only 5.3% unchanged by the kidney. About 60% of the dose was recovered in urine mainly as conjugated PCP and conjugated tetraclorohydroquinone. For both routes of administration, about 10% of the dose was recovered in feces as PCP and its metabolites, which indicates that biliary excretion contributes to total elimination (Reigner et al. 1991)... 

The conjugated-PCP in the bile(5.7 g) collected from 30 goldfish (av. 110 g) exposed to 0.1 ppm PCP for 48 h was isolated by treating the bile with activated charcoal columns, followed by elution with an acetone-ammonia mixture and finally by passing... 

After 15-h exposure to 560 liters of 0.5 ppm PCP, 240 gold-fish(av. 35 g) were transferred to 560 liters of PCP-free water and cultured for 24 h. A conjugated-PCP amounting to 0.36 mmoles was excreted in the water during the 24 h culture period. The conjugate was isolated by a procedure similar to that used for the isolation of the PCP-conjugate from bile, as mentioned above. 

The amounts of free- and conjugated-PCP contained in the overflow from the compartment(A), the water in the compartment(B), the urine and the bile collected from the fish were also determined. 

Table III shows the amounts of free- and conjugated-PCP excreted from each of the biliary, renal and branchial routes.

Free-PCP (pinole) Conjugated-PCP (jjmole) Free-PCP (yimole) Conj ugated-PCP (pmole)... 

Kobayashi K, Akitake H, Tomiyama T (1970) Studies on the metabolism of pentachlorophenate, a herbicide, in aquatic organisms — III. Isolation and identification of a conjugated PCP yielded by a shell-fish. Tapes philippinarum. Bull Jpn Soc Sci Fish 36 103-108 Koivusaari U, Lindstrom-Seppa P, Lang M, Harri M, Hanninen O (1980) Occurrence of cytochrome P-450 in certain fresh water species in northern Europe. In Gustafsson JA, Carlstedt-Duke J, Mode A, Rafter J (eds) Biochemistry, biophysics and regulation of cytochrome P-450. Elsevier/North-Holland, Amsterdam, pp 455-458... 

Harano and colleagues [48] found that the reactivity of the Diels-Alder reaction of cyclopentadienones with unactivated olefins is enhanced in phenolic solvents. Scheme 6.28 gives some examples of the cycloadditions of 2,5-bis-(methoxycar-bonyl)-3,4-diphenylcyclopentadienone 45 with styrene and cyclohexene in p-chlorophenol (PCP). Notice the result of the cycloaddition of cyclohexene which is known to be a very unreactive dienophile in PCP at 80 °C the reaction works, while no Diels-Alder adduct was obtained in benzene. PCP also favors the decarbonylation of the adduct, generating a new conjugated dienic system, and therefore a subsequent Diels-Alder reaction is possible. Thus, the thermolysis at 170 °C for 10 h of Diels-Alder adduct 47, which comes from the cycloaddition of 45 with 1,5-octadiene 46 (Scheme 6.29), gives the multiple Diels-Alder adduct 49 via decarbonylated adduct 48. In PCP, the reaction occurs at a temperature about 50 °C lower than when performed without solvent, and product 49 is obtained by a one-pot procedure in good yield. 

Biotransformation of PCP in mammals occurs via conjugation, reductive dechlorination, hydrolytic dechlorination, and oxidation. In the process, a number of metabolites are formed, some of which are demonstrably toxic (Renner and Hopfer 1990 Jansson and Jansson 1991 Reigner et al. 1991). Metabolites of PCP found in rat urine and identified (acute oral LD50 to mice, in mg/kg BW) include (Renner and Hopfer 1990) ... 

Highest residues were in liver, kidney, and lungs in milk, fat fraction contained the greatest amount. Tb 1/2 for absorption was 4.3 h, and for elimination 43 h. Most excretion was by way of urine (76%), then milk and feces (5% each). In urine, PCP was present in the conjugated form (Kinzell et al. 1985)... 

After a single dose, elimination occurred by way of several routes catabolism to tetrachlorohydroquinone excretion of unchanged PCP and its glucuronide conjugate in urine excretion of PCP or its metabolites into bile. More than 90% was eliminated during the rapid phase, the Tb 1/2 being 13-17 h (Braun etal. 1977)... 

The present paper deals with a relation between toxicity and accumulation of chlorophenols in goldfish, Carassius auratus, PCP metabolites and their amounts excreted by the three major routes (branchial, renal and biliary) in the fish, and also with effects of pre-exposure to PCP on PCP-tolerance and on sulfate conjugation with phenol by the liver soluble fraction of the fish. 

Glickman et al. have also reported that PCP-glucuronide is excreted in the bile of rainbow trout exposed to PCP and penta-chloroanisole media(7). The biliary excretion after glucuronide conjugation must be one of general detoxification mechanisms for PCP in fish. 

When goldfish were transferred from PCP-media to PCP-free water, the PCP absorbed by the fish was quickly excreted into surrounding water with a half-life of ca. 10 h(4), mostly in a conjugated-form accompanied with a small amount of free-form(8). 

The isolated conjugate was identified as pentachlorophenyl-sulfate by precipitation with BaCl2, by column and thin-layer chromatography, by UV-absorption spectra, and by determination of the molar ratio of PCP to SO4. PCP-glucuronide which is excreted in bile was not detected(j0. 

A PCP-conjugate excreted by short-necked clam, Tapes philip-pinarum. into surrounding water has been also identified as the sulfate-conjugate(9). 

As shown in Table IV (12), a considerable amount of the PCP-glucuronide in bile was hydrolyzed by the intestinal mucus. This indicates that the glucuronide conjugation plays an important role in reduction of the concentration of free-PCP in the fish body, but not in elimination of PCP from the fish body as compared with the sulfate conjugation, because the PCP released from the glucuronide in the intestine must be reabsorbed there. 

Effect of Pre-exposure to PCP on PCP-tolerance and Sulfate Conjugation Activity... 

Figure 3. Increase in sulfate conjugation activity of liver-soluble fractions of goldfish exposed to PCP. The fish were exposed to 0.1 ppm PCP every other day...

Figure 3 (13) shows approximately 26 % augmentation in the sulfate conjugation activity of liver soluble fractions of the fish exposed to PCP for 4 days, when compared with the control group. 

The pre-exposure to PCP increased both the PCP-tolerance and the sulfate conjugation activity in goldfish. This suggests that fish have some ability to increase their PCP-tolerance when the fish had been exposed to sublethal PCP-media, and also that the sulfate conjugation activity is an important factor determining the PCP-tolerance of fish. 

Probing the reactions catalyzed by other NRPS domains can be difficult, as the reactants are frequently aminoacyl/peptidyl-S-PCP conjugates. In 2000, Walsh and coworkers reported that aminoacyl-N-acetylcys-teamine thioesters (aminoacyl-SNACs), which mimic PCP-bound substrates, could be used to probe the reactivity and specificity of C domains. Peptidyl and aminoacyl thioesters of NAC can also serve as substrate analogues for other NRPS domains, including TE, Cy, and MTs. ... 

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