Piero Dolara

Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism.

Whether the beneficial effects of pomegranate are due to the ellagitannins or to their microbiota-derived urolithins is not known. Our objectives were to evaluate the effects of pomegranate intake and its main microbiota-derived metabolite urolithin-A (UROA) on colon inflammation and to assess whether UROA is the main anti-inflammatory compound. In addition, the effect of the inflammation on the phenolic metabolism was also explored. Male Fisher rats were fed with 250 mg kg(-1) day(-1) pomegranate extract (PE) or 15 mg kg(-1) day(-1) UROA for 25 days. Dextran sodium sulfate (5%) (DSS) was administered for the five last days and then rats were euthanized. DSS is a well-known model of inflammatory bowel disease. Colon tissue damage, microbiota changes, antioxidant status, prostaglandin E(2) (PGE(2)), nitric oxide production, inducible nitric oxide synthase (iNOS), prostaglandin E synthase (PTGES), gene expression (microarrays and RT-PCR) and polyphenol metabolism (LC-MS-MS) were evaluated. Both PE and UROA decreased inflammation markers (iNOS, cycloxygenase-2, PTGES and PGE(2) in colonic mucosa) and modulated favorably the gut microbiota. The G(1) to S cell cycle pathway was up-regulated in both groups. UROA group showed various down-regulated pathways, including that of the inflammatory response. PE, but not UROA, decreased oxidative stress in plasma and colon mucosa. Only UROA preserved colonic architecture. The normal formation of urolithins in PE-fed rats was prevented during inflammation. Our results suggest that UROA could be the most active anti-inflammatory compound derived from pomegranate ingestion in healthy subjects, whereas in colon inflammation, the effects could be due to the nonmetabolized ellagitannin-related fraction.

Polycyclic aromatic hydrocarbon contamination in the Italian diet

The content of total and carcinogenic polycyclic aromatic hydrocarbons (PAHs) in Italian foods was measured. The highest levels of PAHs were found in pizza baked in wood-burning ovens and barbecued beef and pork. Relatively high levels were also found in beet greens and squash, apples and bread, fried beef, pork and rabbit, cured meats and chocolate. Conversely low levels were detected in potatoes and cooked fish, beverages and eggs. The daily intake of total and carcinogenic PAHs also was calculated by multiplying the average consumption of each food by its mean concentration of PAHs. Cereal and milk products, meat, vegetables and fruits were the highest contributors to total PAH intake, since these products are the most important dietary components in Italy. The calculated total dietary PAH intake was 3 micrograms/day per person. The calculated intake of carcinogenic PAHs was 1.4 microgram/day per person. The dietary intake of PAHs was high compared with the calculated respiratory intake (370 ng/day) owing to polluted city air in Italy. These results confirm that food is the major source of human exposure to PAHs, due in particular to the high consumption of contaminated cereal products.

Effect of a low dose of dietary resveratrol on colon microbiota, inflammation and tissue damage in a DSS-induced colitis rat model.

The naturally occurring polyphenol resveratrol has been acknowledged with health-beneficial properties. Most of the studies dealing with its in vivo effects assay huge doses, not representative from a dietary point of view. Our aim was to ascertain whether resveratrol can exert anti-inflammatory activity in vivo at an attainable dietary dose. Rats were fed with 1 mg of resveratrol/kg/day (a human equivalent dose) for 25 days, and in the last 5 days, 5% dextran sulfate sodium (DSS) was administered to induce colitis. Effects on colon tissue damage, gut microbiota, reactive oxygen species, inflammatory markers and nitric oxide production as well as gene expression profile with microarrays were evaluated. Resveratrol increased lactobacilli and bifidobacteria as well as diminished the increase of enterobacteria upon DSS treatment. Resveratrol significantly protected the colonic mucosa architecture, reduced body weight loss, diminished the induced anemia and reduced systemic inflammation markers, colonic mucosa prostaglandin E(2), cycloxygenase-2, prostaglandin E synthase and nitric oxide levels. In addition, the expression of 2,655 genes in distal colon mucosa related to important pathways was varied. These results reinforce the concept of resveratrol as a dietary beneficial compound in intestinal inflammation at doses possibly attainable with resveratrol-enriched nutraceuticals.

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes.

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.

Rectal proliferation and polyp occurrence in patients with familial adenomatous polyposis after sulindac treatment

BACKGROUND/AIMS Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), decreases the occurrence of polyps in patients with familial adenomatous polyposis (FAP). The effects of colectomy with ileorectal anastomosis (IRA) and sulindac treatment on rectal mucosa proliferation and polyp occurrence were examined in patients with FAP. METHODS The number and size of rectal polyps were measured with colonoscopy. The labeling index, the percentage of labeled cells per crypt compartment, was assessed in rectal biopsy specimens with [3H]thymidine incorporation and autoradiography in 6 non-IRA and 14 IRA patients before and after treatment with 200 mg of sulindac/day for 60 days. RESULTS The IRA patients had a lower labeling index and a decrease in the percentage of labeled cells in the upper compartment of the crypt (P < 0.01) relative to non-IRA subjects. Sulindac did not influence the labeling index and the distribution of labeled cells along the crypt. On the contrary, a dramatic decrease in the size and number of polyps was observed after sulindac treatment (P < 0.001). CONCLUSIONS The persistence of a abnormal mucosal proliferation after sulindac therapy, in spite of the reduction of polyp number, suggests caution in assuming a lower risk of rectal cancer in patients with FAP.

Effect of natural phenolic acids on DNA oxidation in vitro.

We examined the antioxidant activity of the following natural phenolic compounds present in food: 3-OH-benzoic acid (3-OH-BA); 4-OH-benzoic acid (4-OH-BA); 2,3-dihydroxybenzoic acid (2,3-diOH-BA); 3,4-dihydroxybenzoic acid (3,4-diOH-BA or protocatechuic acid); ferulic acid; caffeic acid; and 2-coumaric, 3-coumaric and 4-coumaric acids. We measured the inhibitory effect of these compounds on iron-dependent oxidative DNA damage in vitro [incubating herring sperm DNA with Fe(III)/GSH] or using cumene hydroperoxide (CumOOH) as a free-radical generating system; we also studied the interaction of these phenols with Fe(II) or Fe(III) spectrophotometrically. Among the tested compounds, 2,3-diOH-BA, 3,4-diOH-BA and caffeic acid interacted with Fe(II) and showed a potent inhibitory effect on iron-induced oxidative DNA damage. CumOOH-induced DNA oxidation was not modified by these compounds. On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids did not interact with iron but protected against oxidative DNA damage induced by Fe(III)/GSH and by CumOOH, indicating a direct free-radical scavenging activity of these compounds in both systems. The IC(50)+/-S.E.M. of the three coumaric acids against CumOOH-induced DNA oxidation was 44.2+/-2.0, 54.7+/-2.0 and 33.1+/-1.0 microM, respectively. On the contrary, 3-OH-BA and 4-OH-BA did not have scavenging activity and 3-OH-BA actually enhanced oxidative DNA damage. In conclusion, some natural phenolic acids, commonly present in food, have interesting protective activity against DNA oxidation in vitro and deserve further consideration as effective antioxidants in vivo.

Effect of taurine on calcium kinetics of guinea-pig heart

Abstract Guinea-pig hearts, perfused with Tyrode containing 8 mM taurine, show a less marked decrease of contractile force when washed out with calcium-free Tyrode than control hearts. The effects of taurine cardiac contractility are explained by its interference with calcium kinetics. Total calcium content is increased in taurine-treated hearts, and greater amounts of calcium are washed out from calcium exchanging compartments, analyzed according to the model of Bayley et al. (1968). Most of the additional calcium retained by taurine-treated hearts is bound to some tissue structure, and can be partially mobilized when the concentration of easily exchanged calcium falls. The hypothesis is advanced that taurine increases the affinity of some cell structure for calcium.

NK1-receptors mediate the proliferative response of human fibroblasts to tachykinins.

1 The effect of synthetic tachykinin selective receptor agonists was studied on the growth of cultured human skin fibroblasts (HF). 2 Human fibroblasts were grown in serum‐free conditions in the presence of natural tachykinins (substance P and neurokinin A) and of three synthetic agonists, [β‐Ala4, Sar9, Met(O2)11]‐SP(4–11), [β‐Ala8]‐NKA(4–10) and [MePhe7]‐NKB selective for NK1‐, NK2‐ and NK3‐receptors respectively. Cell proliferation was measured by percentage increase in cell number and by [3H]‐thymidine uptake following 48 h exposure to agents compared to baseline condition. 3 Neurokinin A (NKA) and substance P (SP) significantly increased cell proliferation the threshold concentrations being 10−12 and 10−11 m, respectively. Addition of thiorphan to culture conditions enhanced the effect of SP but not of NKA. 4 The selective NK1‐receptor agonist produced a dose‐dependent increase in cell proliferation as judged by total cell number and [3H]‐thymidine uptake. No significant effect was observed with NK2‐ and NK3‐receptor agonists. 5 These data indicate that the effect of SP on fibroblast proliferation is mediated by interaction with a NK1‐receptor type and local metabolism can interfere with the full expression of this effect of SP on cell proliferation.

NF-κB-dependent anti-inflammatory activity of urolithins, gut microbiota ellagic acid-derived metabolites, in human colonic fibroblasts

Previous studies have reported the anti-inflammatory properties of pomegranate extracts, suggesting that ellagitannins (ET) and ellagic acid (EA) are the main anti-inflammatory compounds. However, both ET and EA are metabolised in vivo by the gut microbiota to yield urolithins (Uro) which can be found in the gut and in systemic bloodstream. The present study was carried out to evaluate the individual effect of EA and their microbiota-derived metabolites Uro on colon fibroblasts upon IL-1beta treatment as an in vitro inflammation model. Uro-A and Uro-B (10 microm) inhibited PGE2 production (85 and 40 %, respectively) after IL-1beta stimulation, whereas EA did not show any effect. Uro-A, but not Uro-B, down-regulated cyclo-oxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) mRNA expression and protein levels. Both Uro inhibited NF-kappaB translocation to nucleus. Slight but significant effects were found in the activation of mitogen-activated protein kinase (MAPK) pathways. Uro-A lowered c-Jun N-terminal kinase phosphorylation state, and both Uro inhibited p38 activation. No metabolites derived from Uro or EA were found in the cell media upon incubation of EA or Uro with the cells, and only traces of the compounds were found inside the cells. The present results suggest that Uro, mainly Uro-A, are the main compounds that are responsible for the pomegranate anti-inflammatory properties. The mechanism of action implicated seems to be via the inhibition of activation of NF-kappaB and MAPK, down-regulation of COX-2 and mPGES-1 expressions, and consequently,via the reduction of PGE2 production. Taking into account that Uro did not enter the cells, a competitive binding for IL-1beta membrane receptor cannot be discarded.

Polycyclic aromatic hydrocarbons air levels in Florence, Italy, and their correlation with other air pollutants.

Benzo(a)pyrene [B(a)P] air levels were measured in Florence (Italy) in the period 1992-2001. For the period 1999-2000 seven polycyclic aromatic hydrocarbons (PAH) (benzo(a)anthracene, crysene, benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene, dibenzo(a,h)anthracene (DBA) and benzo(g,h,i)perylene (BGP)), were measured in the air in four different sites (one with heavy traffic (A), one in a park (B), one in a residential area (C) and one in a hill area (D)). B(a)P levels were elevated in 1992-1998 (maximum average value of winter months: 5.8 ng/ m3) but a decreasing trend was observed in the following years, probably due to improvement in vehicle emissions. The sum of PAH in the air in the period 1999-2000 was about one order of magnitude lower in the hill site (D) relative to the urban sites, and residential areas (B and C) had values 2.5-3 times lower compared to site A with a heavy traffic. PAH concentrations decreased in the warmer seasons of 2000 in all sites. A negative correlation was found between PAH levels and ozone. A positive correlation with carbon monoxide (CO) (r = 0.862, P < 0.001) and low B(a)P/BGP ratios, ranging from 0.44 to 0.51, indicated that vehicular traffic was the major PAH source in all monitored sites. Using B(a)P(TEF) values (toxic equivalency factors) for evaluating the biological activity of PAH, we found that the highest PAH contributors in terms of potential air carcinogenic activity were B(a)P and DBA. Therefore, in addition to B(a)P, DBA concentration should be considered in the evaluation of air quality in terms of PAH contamination.