Genetic and other sources of variation in the activity of serum paraoxonase/diazoxonase in humans: consequences for risk from exposure to diazinon.

Diazinon is the only organophosphorus insecticide that is currently approved for use in sheep dip in the UK. Reports that some individuals may be genetically more susceptible to possible chronic adverse health effects, due to variations in PON1 activity, are complicated by the reliability of activity measurements. In the present study, the influence of three polymorphisms of PON1 on serum diazoxonase activity was investigated in 85 healthy volunteers. Serum activity was assessed in as close to physiological conditions as possible (at pH 7.4, 150 mM NaCl and 37 degrees C with 50 microM diazoxon as substrate) and by quantifying pyrimidinol formation using high-performance liquid chromatography. PON1 genotypes were determined by the polymerase chain reaction and restriction enzyme digestion. For PON1 Q192R, individuals with the RR genotype had the highest serum diazoxonase activity, in contrast to some previous reports where activity was determined under less physiological conditions. Activity was slightly reduced in individuals with the QR genotype and activity was reduced even further in those with the QQ genotype. For PON1 L55 M, there was a significant decrease in mean enzyme activity from LL>LM>MM genotypes. The promoter polymorphism PON1 -108 C/T had only a slight effect on activity. Overall, intragenotype variation in PON1 activity was appreciably greater than the mean intergenotype differences. In conclusion, although there is a wide variation in activity in individuals both within and between genotypes, those individuals with a combination of Q and M alleles generally have a lower ability to detoxify diazoxon, which implies a potentially greater susceptibility to toxicity from diazinon.

Effect of flavonoids and vitamin E on cyclooxygenase-2 (COX-2) transcription.

Cyclooxygenase-2 (COX-2)-catalysed synthesis of prostaglandin E2 plays a key role in inflammation and its associated diseases, such as cancer and cardiovascular disease. There are numerous reports demonstrating that flavonoids inhibit COX-2 activity. However, transcriptional regulation of COX-2 can also be important. Nobiletin, amentoflavone, quercetin, quercetin penta-acetate, flavone, resveratrol, apigenin, chrysin, kaempferol, galangin, and genistein have been reported to modulate COX-2 transcription in a wide variety of systems. Here, we briefly review the literature on regulation of COX-2 transcription by flavonoids, and report some new preliminary data on Vitamin E and quercetin conjugates. Quercetin, quercetin 3-glucuronide, quercetin 3'-sulfate and 3'methylquercetin 3-glucuronide reduced COX-2 mRNA expression in both unstimulated and interleukin-1beta stimulated colon cancer (Caco2) cells. Quercetin and quercetin 3'-sulfate, unlike quercetin 3-glucuronide and 3'methylquercetin 3-glucuronide, also inhibited COX-2 activity. In contrast, tocopherols (alpha-tocopherol, alpha-tocopherol acetate, and gamma-tocopherol at 10microM) did not affect COX-2 mRNA expression in unstimulated Caco2 cells. However, the tocopherols inhibited COX-2 activity showing that the tocopherols act post-transcriptionally on activity, whereas quercetin and some quercetin conjugates affect both the transcription and activity of COX-2. Flavonoid modulation of COX-2 transcription may therefore be an important mechanism in anti-carcinogenesis.

Quercetin metabolites downregulate cyclooxygenase-2 transcription in human lymphocytes ex vivo but not in vivo.

Flavonoids have the potential to modulate inflammation by inhibition of cyclooxygenase-2 (COX-2) transcription. In this study, we compared the effect of the human flavonoid plasma metabolites (quercetin 3'-sulfate, quercetin 3-glucuronide and 3'-methylquercetin 3-glucuronide) on expression of COX-2 mRNA in human lymphocytes ex vivo using TaqMan real-time RT-PCR. We show that the flavonoid quercetin metabolites as detected in human plasma at physiologically significant concentrations inhibit COX-2 expression in human lymphocytes ex vivo. To examine the effect in vivo, we measured COX-2 mRNA levels in 8 subjects (5 men and 3 women) participating in a 3-way, single-blind, randomized crossover study after consumption of a single meal of white, low-quercetin onions, compared with yellow, high-quercetin onions. After consumption of high-quercetin onions, quercetin conjugates were detected in plasma (up to a maximum concentration of 4 micro mol/L at approximately 1 h). However, the expression of COX-2 mRNA in lymphocytes was unchanged by the consumption of high-quercetin onions compared with the low-quercetin group. The results show that a single high dose of the flavonoid quercetin from onions does not change COX-2 mRNA expression in human lymphocytes in vivo even though this change occurred in vitro and ex vivo.

Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo.

For the first time the human intestinal effective permeability, estimated from the luminal disappearance and intestinal metabolism of phytochemicals, sulforaphane and quercetin-3,4'-glucoside, as well as the simultaneous changes in gene expression in vivo in enterocytes, has been studied in the human jejunum in vivo (Loc-I-Gut). Both compounds as components of an onion and broccoli extract could readily permeate the enterocytes in the perfused jejunal segment. At the physiologically relevant, dietary concentration tested, the average effective jejunal permeability (Peff) and percentage absorbed (+/- S.D.) were 18.7 +/- 12.6 x 10-4 cm/s and 74 +/- 29% for sulforaphane and 8.9 +/- 7.1 x 10-4 cm/s and 60 +/- 31% for quercetin-3,4'-diglucoside, respectively. Furthermore, a proportion of each compound was conjugated and excreted back into the lumen as sulforaphane-glutathione and quercetin-3'-glucuronide. The capacity of the isolated segment to deconjugate quercetin from quercetin-3,4'-diglucoside during the perfusion was much higher than the beta-glucosidase activity of the preperfusion jejunal contents, indicating that the majority (79-100%) of the beta-glucosidase capacity derives from the enterocytes in situ. Simultaneously, we determined short-term changes in gene expression in exfoliated enterocytes, which showed 2.0 +/- 0.4-fold induction of glutathione transferase A1 (GSTA1) mRNA (p < 0.002) and 2.4 +/- 1.2-fold induction of UDP-glucuronosyl transferase 1A1 (UGT1A1) mRNA (p < 0.02). The changes in gene expression were also seen in differentiated Caco-2 cells, where sulforaphane was responsible for induction of GSTA1 and quercetin for induction of UGT1A1. These results show that food components have the potential to modify drug metabolism in the human enterocyte in vivo very rapidly.

Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism.

Quercetin-3- and quercetin-7-glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10(6) cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10(6) cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous beta-glucuronidase followed by sulfation to quercetin-3'-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10(6) cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10(6) cells, over 48 hr). In contrast, quercetin-4'-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by beta-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol-O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3'-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10(6) cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin glucuronides and that human endogenous beta-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.