The Comparative Metabolism of Xenobiotics

Despite the growing concern on the presence of pharmaceutically active compounds in the environment, few studies have been conducted on their metabolism in marine organisms. In this study, a non-targeted strategy based on the generation of chemical profiles generated by liquid chromatography combined with high resolution mass spectrometry was used to highlight metabolite production by the Mediterranean mussel (Mytilus galloprovincialis) after diclofenac exposure. This method allowed revealing the production of 13 metabolites in mussel tissues. Three of them were phase I metabolites, including 4′-hydroxy-diclofenac and 5-hydroxy-diclofenac. The remaining 10 were phase II metabolites, including sulfate and amino acids conjugates. Among all of the metabolites highlighted, 5 were reported for the first time in an aquatic organism exposed to diclofenac.

A herbicide containing 2,4-dichlorophenoxyacetic acid (2,4-D) and related chemicals was fed to caterpillars of Eupackardia calleta, and the fate of the substances in the larvae and during further ontogenesis was followed by combined gas chromatography/mass spectrometry. The compounds were found in differing amounts in larval midgut, faeces, fat body/haemolymph, and even in an exocrine secretion produced by integumental glands. Furthermore, they were detected in samples from the resulting adult moths, indicating an intraindividual transfer. Since the individual development of E. calleta was distinctly accelerated by 2,4-D, possible impacts of the herbicide on the life history of the animals in the field are discussed. Based on the chemical data, hypothetical metabolic pathways for 2,4-D in E. calleta larvae are proposed.

Tyrosine β-glucosyltransferase activity in larval tissues of Manduca sexta was determined by reversed-phase HPLC to quantify the product tyrosine glucoside [β-d-glucopyranosyl-O-l-tyrosine, (TG)] formed in incubation mixtures. Synthesis of TG occurred only in fat body preparations, with most of the enzyme activity (95%) in the 15,000 g pellet of homogenates. Other tissues were devoid of activity but did support glucosylation of phenolic substrates other than tyrosine. Activity was greatest with uridine 5′-diphosphoglucose (UDPG) as the glucose donor. However, thymidine 5′-diphosphoglucose (dTDPG) afforded approximately one fourth the amount of tyrosine glucosylation provided by UDPG. Magnesium ions at concentrations up to 15 mM stimulated activity, whereas Ca²⁺ and Mn²⁺ were only slightly stimulatory at 5 mM but progressively inhibited TG synthesis at higher concentrations as did Co²⁺. Maximal rates of glucosylation occurred in the pH range 7.5–9.0. The enzyme was highly specific for tyrosine, because no glucosylation occurred for a number of tyrosine derivatives. Several natural and synthetic mono- and diphenols were found to be weak to moderate inhibitors of the enzyme. In a study of tyrosine β-glucosyltransferase activity during development, no TG synthesis occurred in enzyme preparations from first, second, third, or fourth larval instars, although glucosylation of p-nitrophenol and 4-hydroxycoumarin did occur. Activity was not detected in the fat body of newly ecdysed fifth instars, but low levels were observed 36 h later. The rate of tyrosine glucosylation continued to increase to a peak at 4 days, but then decreased to very low levels after cessation of larval feeding. Low activity was also observed in the pharate adult fat body about 1 day before eclosion. Therefore, tyrosine β-glucosyltransferase activity occurs primarily in the fat body of fifth stadium larvae for synthesis of the pupal cuticle tanning precursor tyrosine glucoside.

Activity of phenol β-glucosyltransferase (PGT) in labial gland and fat body tissue preparations from feeding 5th stadium larval tobacco hornworms, Manduca sexta, towards a variety of plant phenolics was determined by HPLC analysis of the β-glucoside product formed. The enzymes in the 15,000 g fractions of tissue homogenates with uridine 5′-diphosphoglucose as a cofactor showed a wide range of activity in conjugating simple mono-, di-, and triphenols; phenolic acids, aldehydes, and alcohols; and coumarins and flavonoids. Highest specific activity of both labial glan and fat body enzyme preparations occured with diphenol substrates such as catechol, hydroquinone, and vanillin, whereas lowest activities generally were observed with flavonoid substrates. The labial gland PGT had higher specific activity for the simple phenolic substrates than did the fat body enzyme. However, the coumarins and flavonoids were glucosylated at nearly the same rates by both enzyme preparations. Although the functions of PGT in labial glands and fat body are unknown, evidence from this study suggests that they could be important in conjugating a number of potentially toxic plant phenols occuring in the larval diet.

• 1.

  1. Phenol β-glucosyltransferase (PGT) activity in tissues of six species of insects was determined by HPLC with UV/VIS detection of the β-glucoside products.

• 2.

  2. PGT activity was associated mainly with the 15,000 g pellet fraction of tissue homogenates, the fat body generally had the highest specific activity followed by the midgut.

• 3.

  3. PGT activity was greatest with UDPG as a glucose donor, followed by dTDPG and GDPG.

• 4.

  4. A broad range of phenolic compounds, including mono- and diphenols, coumarins and a flavonoid, were glucosylated by insect PGTs. Two generalist herbivores, the grasshopper Melanoplus sanguinipes and the black cutworm Agrotis ipsilon, had the highest PGT activities, whereas other species, with more limited food selection, had lower rates of glucosylation.