Zachary T. Bitzer

Cocoa procyanidins with different degrees of polymerization possess distinct activities in models of colonic inflammation

Procyanidins are available in the diet from sources such as cocoa and grapes. Procyanidins are unique in that they are comprised of repeating monomeric units and can exist in various degrees of polymerization. The degree of polymerization plays a role in determining the biological activities of procyanidins. However, generalizations cannot be made regarding the correlation between procyanidin structure and bioactivity because the size-activity relationship appears to be system dependent. Our aim was to screen fractions of procyanidins with differing degrees of polymerization in vitro for anti-inflammatory activities in models of colonic inflammation. Monomeric, oligomeric and polymeric cocoa procyanidin fractions were screened using cell models of disrupted membrane integrity and inflammation in human colon cells. High-molecular-weight polymeric procyanidins were the most effective at preserving membrane integrity and reducing secretion of interleukin-8 in response to inflammatory stimuli. Conversely, oligomeric procyanidins appeared to be the least effective. These results suggest that polymeric cocoa procyanidins may be the most effective for preventing loss of gut barrier function and epithelial inflammation, which are critical steps in the pathogenesis of metabolic endotoxemia, inflammatory bowel disease and colon cancer. Therefore, further investigations of the potential health-protective benefits of cocoa procyanidins with distinct degrees of polymerization, particularly high-molecular-weight procyanidins, are warranted.

(-)-Epigallocatechin-3-gallate decreases colonic inflammation and permeability in a mouse model of colitis, but reduces macronutrient digestion and exacerbates weight loss.

SCOPE (-)-Epigallocatechin-3-gallate (EGCG) has been reported to have putative health effects including the prevention of inflammation and obesity. Historically, polyphenols have been regarded as antinutritionals and while such effects may be beneficial in obese subjects, they may be deleterious in nutritionally compromised individuals. METHODS AND RESULTS We examined the effect of EGCG in the dextran sulfate sodium (DSS)-treated mouse model of ulcerative colitis. Following induction of colitis, mice were treated with EGCG (3.2 mg/g) as the sole source of drinking fluid for 3 days. EGCG treatment mitigated DSS-induced colon shortening and spleen enlargement. EGCG also decreased colonic protein levels of IL-1β, IL-6, and tumor necrosis factor-α, as well as colonic lipid peroxides compared to DSS-treated controls. We observed that EGCG reduced DSS-induced gastrointestinal permeability. These beneficial effects were offset by enhanced body weight loss in EGCG-treated mice compared to DSS-treated controls. These effects were related to decreased protein and lipid digestion in EGCG-treated mice compared to DSS-treated controls. CONCLUSIONS Our results suggest that although EGCG may exert anti-inflammatory effects, its ability to modulate macronutrient digestion may represent a dose-limiting adverse effect that must be considered in the context of its use for treating inflammatory bowel disease.

Effects of Solvent and Temperature on Free Radical Formation in Electronic Cigarette Aerosols

The ever-evolving market of electronic cigarettes (e-cigarettes) presents a challenge for analyzing and characterizing the harmful products they can produce. Earlier we reported that e-cigarette aerosols can deliver high levels of reactive free radicals; however, there are few data characterizing the production of these potentially harmful oxidants. Thus, we have performed a detailed analysis of the different parameters affecting the production of free radical by e-cigarettes. Using a temperature-controlled e-cigarette device and a novel mechanism for reliably simulating e-cigarette usage conditions, including coil activation and puff flow, we analyzed the effects of temperature, wattage, and e-liquid solvent composition of propylene glycol (PG) and glycerol (GLY) on radical production. Free radicals in e-cigarette aerosols were spin-trapped and analyzed using electron paramagnetic resonance. Free radical production increased in a temperature-dependent manner, showing a nearly 2-fold increase between 100 and 300 °C under constant-temperature conditions. Free radical production under constant wattage showed an even greater increase when going from 10 to 50 W due, in part, to higher coil temperatures compared to constant-temperature conditions. The e-liquid PG content also heavily influenced free radical production, showing a nearly 3-fold increase upon comparison of ratios of 0:100 (PG:GLY) and 100:0 (PG:GLY). Increases in PG content were also associated with increases in aerosol-induced oxidation of biologically relevant lipids. These results demonstrate that the production of reactive free radicals in e-cigarette aerosols is highly solvent dependent and increases with an increase in temperature. Radical production was somewhat dependent on aerosol production at higher temperatures; however, disproportionately high levels of free radicals were observed at ≥100 °C despite limited aerosol production. Overall, these findings suggest that e-cigarettes can be designed to minimize exposure to these potentially harmful products.

Effect of Flavoring Chemicals on Free Radical Formation in Electronic Cigarette Aerosols

Background: Flavoring chemicals, or flavorants, have been used in electronic cigarettes (e‐cigarettes) since their inception; however, little is known about their toxicological effects. Free radicals present in e‐cigarette aerosols have been shown to induce oxidative stress resulting in damage to proliferation, survival, and inflammation pathways in the cell. Aerosols generated from e‐liquid solvents alone contain high levels of free radicals but few studies have looked at how these toxins are modulated by flavorants. Objectives: We investigated the effects of different flavorants on free radical production in e‐cigarette aerosols. Methods: Free radicals generated from 49 commercially available e‐liquid flavors were captured and analyzed using electron paramagnetic resonance (EPR). The flavorant composition of each e‐liquid was analyzed by gas chromatography mass spectroscopy (GCMS). Radical production was correlated with flavorant abundance. Ten compounds were identified and analyzed for their impact on free radical generation. Results: Nearly half of the flavors modulated free radical generation. Flavorants with strong correlations included &bgr;‐damascone, &dgr;‐tetradecalactone, &ggr;‐decalactone, citral, dipentene, ethyl maltol, ethyl vanillin, ethyl vanillin PG acetal, linalool, and piperonal. Dipentene, ethyl maltol, citral, linalool, and piperonal promoted radical formation in a concentration‐dependent manner. Ethyl vanillin inhibited the radical formation in a concentration dependent manner. Free radical production was closely linked with the capacity to oxidize biologically‐relevant lipids. Conclusions: Our results suggest that flavoring agents play an important role in either enhancing or inhibiting the production of free radicals in flavored e‐cigarette aerosols. This information is important for developing regulatory strategies aimed at reducing potential harm from e‐cigarettes. HIGHLIGHTSFlavoring chemicals can modulate the production of free radicals in e‐cigarettes.Citral, dipentene, ethyl maltol, linalool, and piperonal all promote radical formation.Ethyl vanillin dose‐dependently inhibits radical delivery.Flavorants can modulate lipid peroxidation induced by e‐cigarette aerosols.

Effects of Topography-Related Puff Parameters on Carbonyl Delivery in Mainstream Cigarette Smoke

Smoking topography parameters differ substantially between individual smokers and may lead to significant variation in tobacco smoke exposure and risk for tobacco-caused diseases. However, to date, little is known regarding the impact of individual puff parameters on the delivery of many harmful smoke constituents including carbonyls. To examine this, we determined the effect of altering individual puff parameters on mainstream smoke carbonyl levels in machine-smoked reference cigarettes. Carbonyls including formaldehyde, acetaldehyde, crotonaldehyde, propionaldehyde, methyl ethyl ketone (MEK), acrolein, and acetone were determined in cigarette smoke by HPLC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Deliveries of all carbonyls were nearly two-fold greater when cigarettes were smoked according to the more intense Health Canada Intense (HCI) protocol compared to the International Organization of Standardization (ISO) method, consistent with the two-fold difference in total puff volume between methods (ISO: 280-315 mL; CI: 495-605 mL). When individual topography parameters were assessed, changes in puff volume alone had the greatest effect on carbonyl delivery as predicted with total carbonyls being strongly correlated with overall puff volume (r2: 0.52-0.99) regardless of how the differences in volume were achieved. All seven of the carbonyls examined showed a similar relationship with puff volume. Minor effects on carbonyl levels were observed from vent blocking and changing the interpuff interval, while effects of changing puff duration and peak flow rate were minimal. Overall, these results highlight the importance of considering topography, especially puff volume, when the toxicant delivery and potential exposure smokers receive are assessed. The lack of an impact of other behaviors, including puff intensity and duration independent of volume, indicate that factors such as temperature and peak flow rate may have minimal overall effects on carbonyl production and delivery.

Variation in Free Radical Yields from U.S. Marketed Cigarettes

Free radicals in tobacco smoke are thought to be an important cause of smoking-induced diseases, yet the variation in free radical exposure to smokers from different brands of commercially available cigarettes is unknown. We measured the levels of highly reactive gas-phase and stable particulate-phase radicals in mainstream cigarette smoke by electron paramagnetic resonance (EPR) spectroscopy with and without the spin-trapping agent phenyl-N-tert-butylnitrone (PBN), respectively, in 27 popular US cigarettes and the 3R4F research cigarette, machine-smoked according to the FTC protocol. We find a 12-fold variation in the levels of gas-phase radicals (1.2 to 14 nmol per cigarette) and a 2-fold variation in the amounts of particulate-phase radicals (44 to 96 pmol per cigarette) across the range of cigarette brands. Gas and particulate-phase radicals were highly correlated across brands (ρ = 0.62, p < 0.001). Both radicals were correlated with TPM (gas-phase: ρ = 0.38, p = 0.04; particulate-phase: ρ = 0.44, p = 0.02) and ventilation (gas- and tar-phase: ρ = -0.58, p = 0.001), with ventilation explaining nearly 30% of the variation in radical levels across brands. Overall, our findings of significant brand variation in free radical delivery under standardized machine-smoked conditions suggest that the use of certain brands of cigarettes may be associated with greater levels of oxidative stress in smokers.

A Survey of Nicotine Yields in Small Cigar Smoke: Influence of Cigar Design and Smoking Regimens

Introduction Although the popularity of small cigar brands that resemble cigarettes, including both little cigars (LC) and filtered cigars (FC), has been on the rise, little is known about the delivery of nicotine from these products. Our objective was to determine the nicotine yields of small cigars in comparison to cigarettes. Methods Nicotine yields from LC, FC, and 3R4F and 1R6F research cigarettes were determined from mainstream smoke generated on a smoking machine under the International Organization of Standardization (ISO) and Canadian Intense (CI) methods. Market characteristics (price and package label) and physical features (filter ventilation, product weight and filter weight, product length, and diameter) were also determined for eight brands of small cigars. Results Nicotine yields in small cigars averaged 1.24 and 3.49 mg/unit on ISO and CI regimens, respectively, compared with 0.73 and 2.35 mg/unit, respectively, for the research cigarettes. Nicotine yields per puff were similar between small cigars and cigarettes. We also found that FC did not differ from LC in nicotine yields. FC and LC differ from each other in many physical design features (unit weight, filter weight, and filter length), but are similar in others (unit length, diameter, and filter ventilation). Conclusions Nicotine delivery from small cigars is similar to or greater than that from cigarettes. Thus, for future research and regulatory purposes, standard definitions need to be developed for small cigars, and FC and LC should be evaluated as separate entities. Implications Small cigars are similar to cigarettes in their design and use. Although nicotine yields per puff were similar between products, small cigars delivered substantially higher amounts of nicotine per unit than cigarettes. These findings support the growing body of evidence to justify regulating all small cigars, including LC and FC in a similar fashion as cigarettes.

Soy protein concentrate mitigates markers of colonic inflammation and loss of gut barrier function in vitro and in vivo

Whereas a number of studies have examined the effects of soy isoflavones and tocopherols on colonic inflammation, few have examined soy protein. We determined the radical scavenging and cytoprotective effects of soy protein concentrate (SPC) in vitro and its anti-inflammatory effects in dextran sulfate sodium (DSS)-treated mice. Cotreatment with SPC protected Caco-2 human colon cells from H2O2-induced cell death and mitigated intracellular oxidative stress. Treatment of differentiated Caco-2 cells with SPC blunted DSS-induced increases in monolayer permeability. Pepsin/pancreatin-digested SPC had reduced radical scavenging activity, but retained the monolayer protective effects of SPC. In vivo, 1.5% DSS caused body weight loss, colon shortening, and splenomegaly in CF-1 mice. Co-treatment with 12% SPC mitigated DSS-induced body weight loss and splenomegaly. DSS increased colonic interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 expression. The levels of these markers were significantly lower in mice co-treated with SPC. SPC prevented DSS-mediated reductions in colonic glucagon-like peptide 2 levels, suggesting that SPC can prevent loss of gut barrier function, but no significant effect on claudin 1 and occludin mRNA levels of was observed. SPC-treated mice had lower colonic mRNA expression of toll-like receptor 4 and nucleotide-binding oligomerization domain-containing protein-like receptor family, pyrin domain containing protein 3 (NLRP3), and lower caspase-1 enzyme activity than DSS-treated mice. In summary, SPC exerted antioxidant and cytoprotective effects in vitro and moderated the severity of DSS-induced inflammation and loss of gut barrier function in vivo. These effects appear to be mediated in part through reduced NLRP3 expression and caspase 1 activity.

Little Cigars, Filtered Cigars, and their Carbonyl Delivery Relative to Cigarettes

Abstract Introduction Little cigars and filtered cigars are currently growing in popularity due to their low cost and wide variety of flavors while retaining an appearance similar to cigarettes. Given the health consequences associated with cigarette use, it is important to understand the potential harm associated with these similar products. This includes the potential harm associated with carbonyls (eg, acetaldehyde, acrolein, formaldehyde, etc.), an important class of toxicants and carcinogens in tobacco smoke. Our objective was to determine the carbonyl levels in mainstream smoke from little and filtered cigars compared to cigarettes. Methods We examined two brands each of little cigars and filtered cigars, as well as two research cigarettes for carbonyl delivery using the International Organization of Standards (ISO) and the Health Canada Intense (HCI) machine-smoking protocols. Results On a per puff basis, the levels of five of the seven carbonyls were higher from little cigars than filtered cigars and cigarettes (ISO: 56–116%; HCI: 39–85%; p < .05). On a per unit basis, most carbonyl levels were higher from both cigar types than cigarettes using the ISO method (ISO: 51–313%; p < .05) whereas only filtered cigars were higher using the HCI method (HCI: 53–99%; p < .05). Conclusion These findings suggest that cigar smokers can be exposed to higher levels of carbonyls per cigar than cigarette smokers per cigarette. Implications These data will increase our understanding of the relative harm from carbonyl exposure from little and filtered cigars both for cigar-only smokers and the cumulative harm among the growing population of cigarette–cigar multi-product smokers.

Free Radical, Carbonyl, and Nicotine Levels Produced by Juul Electronic Cigarettes

Introduction Free radicals and carbonyls produced by electronic cigarettes (e-cigs) have the potential to inflict oxidative stress. Recently, Juul e-cigs have risen drastically in popularity; however, there is no data on nicotine and oxidant yields from this new e-cig design. Methods Aerosol generated from four different Juul flavors was analyzed for carbonyls, nicotine, and free radicals. The e-liquids were analyzed for propylene glycol (PG) and glycerol (GLY) concentrations. To determine the effects of e-liquid on oxidant production, Juul pods were refilled with nicotine-free 30:70 or 60:40 PG:GLY with or without citral. Results No significant differences were found in nicotine (164 ± 41 µg/puff), free radical (5.85 ± 1.20 pmol/puff), formaldehyde (0.20 ± 0.10 µg/puff), and acetone (0.20 ± 0.05 µg/puff) levels between flavors. The PG:GLY ratio in e-liquids was ~30:70 across all flavors with GLY being slightly higher in tobacco and mint flavors. In general, when Juul e-liquids were replaced with nicotine-free 60:40 PG:GLY, oxidant production increased up to 190% and, with addition of citral, increased even further. Conclusions Juul devices produce free radicals and carbonyls, albeit, at levels substantially lower than those observed in other e-cig products, an effect only partially due to a low PG:GLY ratio. Nicotine delivery by these devices was as high as or higher than the levels previously reported from cigarettes. Implications These findings suggest that oxidative stress/damage resulting from Juul use may be lower than that from cigarettes or other e-cig devices; however, the high nicotine levels are suggestive of a greater addiction potential.