Heat-not-burn technology affects plasma testosterone levels and markers of inflammation, oxidative stress in the testes of rats

Abstract

Introduction: Heating tobacco products (HTPs) are advanced electronic cigarette models. Classified by the FDA as a modified-risk tobacco product and can be used as part of efforts to quit smoking. Using heat-not-burn (HnB) technology, these devices heat tobacco avoiding complete combustion. Although the levels of toxicants in the mainstream are significantly lower than those observed in tobacco smoke, some recent studies have raised concerns about potential health risks associated with their use, particularly regarding their effects on male gonadal function, which remain largely unexplored.

Methods: Adult male Sprague-Dawley rats were exposed, whole body, 5 days/week for 4 weeks to HnB mainstream.

Results: The expression of the cell cycle regulators Bax/Bcl-2 ratio is not affected, along with no changes in p-38. On the other hand, an increase in oxidative stress markers, including those associated with DNA damage, was observed in exposed animals, along with the induction of NF-kB dependent pro-inflammatory mediators: TNF-α, IL-1β, IL-6 and COX-2. Furthermore, inactivation of key androgenic enzymes, such as 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase, together with decreased testosterone synthesis suggest a potential impairment of male gonadal function.

Discussion: The results indicate that animals exposed to HnB smoke show higher levels of oxidative stress markers, including those associated with DNA damage, as well as higher levels of pro-inflammatory cytokines. The impairment of some androgenic key enzymes and those related to the activity of seminiferous epithelium, together with the decrease in testosterone levels, suggest an impairment of gonadal function through the alteration of some cellular pathways typically associated with tobacco consumption.

Keywords: DNA damage; heat-not-burn; inflammation; oxidative stress; testis.

FIGURE 1

HnB smoke exposure increases oxidative stress markers in rat testis. The content of radical species, here measured with the DCFH-DA assay, is significantly higher in samples from exposed animals compared to controls (A). HnB group shows an increase in MDA (B) and carbonyls (C) as markers of lipid and protein oxidation, respectively. Higher levels of 8-OHdG (D) as DNA damage marker are here reported in the exposed group compared to controls. Graphs report the means ± SD; *p < 0.05, **p < 0.01, *****p < 0.0001 two-tailed t-test.

FIGURE 2

Animals exposed to HnB smoke show activation of the DNA repair machinery through induction of OGG-1 H2AX and PARP-1 in testes. The base excision repair system OGG-1 (∼47 kDa) (A), ubiquitinated-H2AX (∼25 kDa) (B) and PARP-1 (∼89 kDa) (C) are significantly upregulated in HnB group compared to controls. No significant changes are observed in the expression of xeroderma pigmentosum group C (XPC) (∼26 kDa) (D). The images are representative of two independent experiments. Bars report the means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, Welch’s two-sample two-tailed t-test.

FIGURE 3

HnB smoke exposure induces the NRF2-mediated antioxidant response in rat testis. Samples from animals exposed to HnB show an increase of NRF2 (∼65 kDa) (A), with an upregulation of CAT (B), GSSG-Red (C), SOD (D) and NQO1 (G) enzymatic activities. Immunoblot analysis confirm upregulation of SOD-1 (∼19 kDa) expression (E). GSH-Px (F) showed no significant changes in enzymatic activity. SIRT-1 (H) expression (∼82 kDa) is downregulated. XO (I) expression as marker of OS shows a significant marked upregulation. The images are representative of two independent experiments. Bars represent the means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001, Welch’s two-sample two-tailed t-test.

FIGURE 4

HnB smoking activates NF-κB mediated inflammatory response in rat testis. Exposed animals reported increased phosphorylation of NF-κB (p65 Ser 536) (∼61 kDa) (A), along with the upregulation of TNF-α (∼25 kDa) cleaved form (∼17 kDa) (B), IL-1β (∼17 kDa) (C), IL-6 (∼26 kDa) (D). Here, no significant changes in IL-8 expression (∼11 kDa) were observed (E). Inducible COX-2 (∼50 kDa) (F) was significantly higher in HnB group compared to controls. The images are representative of two independent experiments. Bars represent the means ± SD; *p < 0.05, **p < 0.01, Welch’s two-sample two-tailed t-test.

FIGURE 5

Effects of HnB smoke exposure on steroidogenesis and key functional enzymes of the testis. Data show an impairment of 3β-HSD (A) and 17β-HSD (B) as key enzymes involved in the testosterone synthesis. SDH (C) is downregulated while LDH (D) enzyme activity is significantly higher in HnB group samples compared to controls. Plasma testosterone levels measured here by ELISA are lower in exposed animals than controls (E). The images are representative of two independent experiments. Bars represent the means ± SD; *p < 0.05, ***p < 0.001, Welch’s two-sample two-tailed t-test.

FIGURE 6

No significant changes were recorded for the Bax (∼21 kDa)/Bcl-2 (∼26 kDa) ratio (A) and for p38 (∼40 kDa) (B). ERK1/2 (44–42∼kDa) was significantly activated by the treatment (C) and a marked up-regulation of c-MYC expression (∼49 kDa) was obtained in exposed animals (D). The images are representative of two independent experiments. Bars represent the means ± SD; **p < 0.01; ****p < 0.0001, two-tailed t-test.

FIGURE 7

HnB smoke exposure boosts cytochrome-linked monooxygenase (CYPs) involved in PAH metabolism in rat testis. Data show an induction of CYP1A-dependent monooxygenases, activating aromatic amines, dioxins and PAHs (A) and the relative increase of protein expression (∼63 kDa) (B). CYP2B1/2 (∼66 kDa) (also activating bupropion smoking-cessation drug) (C); CYP2A 1/2 (∼50 kDa) (activating alcohol, nitrosamines, benzene, acetone, acrylamide) (D). The images are representative of two independent experiments. Bars represent the means ± SD; *p < 0.05, ***p < 0.001, two-tailed t-test.

FIGURE 8

Schematic representation of the main effects of HnB mainstream exposure on rat testicular function. HnB smoke exposure increases the production of reactive oxygen species, leading to oxidative DNA damage and the activation of DNA repair enzymes, as well as the activation of NRF2 and NF-kB signaling pathways. These alterations, together with the dysregulation of some androgenic enzymes and the decrease of testosterone biosynthesis, may contribute to the impairment of testicular function.