National Trends in Tobacco Smoke Exposure and its Disruption on Vitamin D Levels Among U.S. Population, 2001-2014

Background: Persuasive evidence suggests that tobacco smoking is endocrine-disrupting and may interfere with vitamin D (VD) endocrine systems, but supporting research is limited and results vary greatly. Methods: Data from the National Health and Nutrition Examination Survey, 2001-2014, was used to evaluate the trends in tobacco smoke exposure among U.S. general participants aged ≥ 3 yr (n=49338). We examined the linear association between serum cotinine and 25(OH)D concentrations, as well as relationship between tobacco smoke exposure categories (active, passive, non-smoking) with VD status (deciency, inadequacy, suciency, intoxication), and assessed whether specic gender, age (3-11, 12-19, 20-59, ≥ 60 yr) or ethnicity/race groups were disproportionately impacted. Results: During 2001-2004, the trends of active smoking rates stabilized between 17.2% to 19.6%. Serum cotinine was signicantly and inversely associated with 25(OH)D in adult participants ( ≥ 20 yr). Tobacco smoke exposure, including both active and passive smoking exposure, was associated with increased risk of VD deciency in adults. Moreover, active smoking of adults was additionally related to enhanced risk of VD inadequacy. These associations showed somewhat gender difference, with consistent and stronger associations observed in female adults. In contrast, effects of tobacco smoke exposure on VD levels were mostly protective or non-signicant among children and adolescents aged 3-19 yr. Conclusion: The percentage of U.S. general population with active smoking exposure stabilized over the 14-yr period and was still high. Tobacco smoke exposure may disrupt vitamin D levels. Our results also provided initial evidence of active smoking exposure on VD intoxication, which needs to be further veried. Implication: Convincing studies have linked tobacco use exposure, including active and passive smoking exposure, to dysfunctional VDES accompanied with declined serum levels of VD metabolites. However, evidence on the association between tobacco smoke exposure and VD status was rather limited, and there were no researches to date that estimated their relationship in children and adolescents. This study analyzed national survey data, to evaluate the national trends in tobacco smoke exposure over a decade, and to comprehensively assess the impacts of tobacco smoke exposure on VD levels across specic gender-, age-and ethnicity/race- groups. The evidence suggests that the prevalence of active smoking exposure stabilized over the 14-yr period and was still high. Moreover, tobacco smoke exposure may disrupt vitamin D levels among general population, with age- and gender- differences observed.


Introduction
Evidence has a rmed the carcinogen, neurotoxic and endocrine-disrupting roles of tobacco use exposure, including both active and passive smoking exposure, in the pathogenesis of a wide range of diseases (Mousavi 2019). The principle component of tobacco products is nicotine. The absorption of nicotine by human body from tobacco is mainly through inhalation via smoke and vaporization exposure; its other routes include oral mucosa absorption via chewing and sni ng, and also through skin absorption ).
Once entering into the body, nicotine is absorbed immediately and rapidly reaches the bloodstream and the brain, leading to an extensive distribution to body tissues. About 70-80% of nicotine is metabolized into cotinine in humans (Benowitz and Jacob 1994).
Substantial declines in smoking rates have been achieved through efforts in public consciousness, education and public policy since 1964 in the United States (National Institute on Drug Abuse, 2020). However, it is worth noticing that smoking has become increasingly appealing to both smokers and non-smokers due to the emergence of electronic cigarettes (e-cigarettes) in the U.S. market since 2007, together with increased avor choices for both traditional and electronic cigarettes. Recent studies have consistently indicated that the tobacco use exposure in the U.S. general population are still high. For example, data accessed from the National Health and Nutrition Examination Survey (NHANES) for 1999-2004 revealed that 9% of adolescents aged 12-19 years and 30% of U.S. adults aged 20 years or older were active smokers according to their serum cotinine values ); meanwhile, differences of age, gender, and ethnicity/race were seen in serum cotinine concentration. Moreover, data from NHANES 2009-2010 reported that about 42% of the U. S. children and adolescents of 3-17 years had serum cotinine concentrations reaching the secondhand smoke exposure levels, with 9% of the teenagers aged 13-17 years reaching the range of active smoking (Nwosu 2018).
Emerging evidence suggests that tobacco use smoke is an endocrine disrupter interfering with Vitamin D endocrine systems (VDES) (Mousavi 2019). Vitamin D (VD) is a fat-soluble hormone and is well known for functions in maintaining bone health and skin barrier. Its nutrigenomic and epigenetic functions have also been demonstrated through links between VD insu ciency or de ciency with various diseases, including tumorigenesis metastasis (Mahamat-Saleh 2020), autoimmune diseases (Sharief 2011; Ahmed 2021), cardiometabolic disorders (Marquina 2018; Pott-Junior 2020), and even worse, coronavirus disease 2019 (COVID-19) risk and severity (Mitchell 2020;Pereira 2020). Substantial evidence also indicates that VD intoxication induces calcium and phosphorus dysregulation, causing damage to tissues and organs (Razzaque 2017). Although VD level was thought to be closely related to sun exposure and dietary intake, independently associated with VD de ciency (VDD) (Nwosu 2018). The NHANES 2001-2006 illustrated that serum cotinine of adults (18-70 years) was associated with lower VD concentration, with results varied by gender and ethnicity/race (Manavi 2015). However, a nationwide study from Korea found no association between cotinine-veri ed smoking and serum VD in adolescents of 10-18 years old (Byun 2017). In addition, these estimations did not consider impacts of exposure on VD intoxication.
To date, there has been limited evidence measuring associations between tobacco use exposure and VD levels, and results varied greatly by time periods, age and gender groups. Therefore, these existing links prompted us to use NHANES data over the entire 14-year period (2001-2014) to: 1) evaluate the trends in tobacco smoke exposure of U.S. general participants, 2) and to comprehensively assess the impacts of tobacco smoke exposure on VD levels across speci c gender-, age-and ethnicity/race-groups to identify susceptibility.

Study population
The National Health and Nutrition Examination Survey, or NHANES, is a nationally ongoing health survey conducted by the Centers for Disease Control and Prevention (CDC)/National Center for Health Statistics (NCHS) since 1959 to monitor the health and nutritional status of the noninstitutionalized U.S. residents (https://www.cdc.gov/nchs/nhanes). Representative participants of all ages were randomly selected through a statistical process using U.S. census information, and were then personally contacted for a home interview concerning sociodemographic characteristics, health history and behaviour. Meanwhile, a one-time speci c health examination was completed based on their age, gender and medication conditions, where biological samples were collected. The NCHS Research Ethics Review Board (ERB) approval and documented consent was obtained from all participants. NHANES released its data for public use in each two-year cycle. Currently, serum cotinine was only available among participants aged ≥ 3 years, thus we included a total of 49338 participants aged ≥ 3 years who had available data on serum cotinine, vitamin D, and sociodemographic covariates (listed below) from seven surveys cycles (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) in the analysis.

Measurement of tobacco use smoke exposure
Tobacco use smoke exposure for NHANES was assessed through the measurement of serum cotinine, a major metabolite of nicotine. Cotinine has been used as a highly speci c and sensitive biomarker in quantifying short-term exposure of tobacco use due to its relatively long half-life (15-20 hours; nicotine: 2 hours), and wide detection in biological uids, including blood, urine, saliva, hair and nails (Benowitz 1996;Akinkugbe 2018).
In each survey cycle, about ninety percent of eligible participants (aged 3 years and older) had cotinine examined in serum in NHANES 2001-2014. Serum cotinine was measured by an isotope-dilution highperformance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (ID HPLC-APCI MS/MS) method. Detailed analytical methodology was available at NHANES website (https://wwwn.cdc.gov/Nchs/Nhanes/2013-2014/COT_H.htm). The lower limit of detection (LLOD in ng/mL) for serum cotinine in each two-year cycle from 2001 to 2014 was 0.015 ng/mL. The results below the LLODs were replaced with the LLODs divided by the square root of two (LLOD/√2). The detection rates of cotinine were summarized in Table 1. According to CDC, nonsmokers exposed to typical levels of passive/secondhand smoke (SHS) have serum cotinine levels of less than 1 ng/mL, with heavy exposure to SHS producing levels in the 1-10 ng/mL range.

Statistical analysis
Because cotinine levels can vary greatly by gender and age, the analyses were strati ed by age group (3-11, 12-19, 20-

Associations of tobacco smoke exposure with VD status by gender-age groups
In children, the estimates for active smokers could not be derived due to small sizes (34, 0.4%). There was no evidence of signi cant associations of passive smoking exposure in children with VD status (Fig. 3 and Supplementary Of note, the associations of tobacco smoke exposure with VD status were stronger in male children and adolescents when compared with females, whereas the association with VD status was stronger in female adults than that in males.

Associations of tobacco smoke exposure with VD status by ethnicity/race and BMI groups
When stratifying by ethnicity/race, passive smoking exposure was positively associated with VD de ciency among participants whose races are Hispanic (OR = 1.70, 95% CI: 1.25, 2.31) and non-Hispanic black (OR = 1.22, 95% CI: 1.04, 1.44). The associations between active smoking and VD de ciency were signi cant in all race groups in Table 3. Speci cally, the association was relatively stronger in non-Hispanic participants when compared with Hispanic and other races. Across ethnicity/race groups, the associations and patterns of active smoking and VD inadequacy were similar to those of VD de ciency. In addition, for Hispanic participants, a strongly positive association was shown between active smoking exposure and VD intoxication (OR = 3.41, 95% CI: 1.72, 6.77). Notes: Estimates were adjusted for gender (categorical), age (continuous), BMI (categorical), PIR (continuous) and NHANES cycle (categorical). "NA" indicates that the analysis could not be derived due to limited cases.
The associations of the active smoking exposure and VD de ciency as well inadequacy were consistently positive for participants across all BMI categories (Table 4). Meanwhile, passive smoking exposure of obese participants was also positively associated with VD de ciency (OR = 1.31, 95% CI: 1.07, 1.59). a Given that there were few cases (1.7%) in the underweight category, underweight and normal weight were combined into one class in the regression analyses.

Results of sensitivity analysis
To exclude the possible in uence of VD intake, we further evaluated the effects of tobacco use exposure on VD status among participants aged 20-59 years (Table 5). Signi cant associations were shown for all types of tobacco smoke exposure and VD de ciency. Notes: Estimates were presented as odd ratios (ORs) and 95% con dence intervals (CIs) and were adjusted for age (continuous), ethnicity/race (categorical), PIR (continuous), BMI (categorical), alcohol use (dichotomous), vigorous and moderate activity (dichotomous), kidney health condition (dichotomous), NHANES cycle (categorical) and VD intake (continuous). Gender was also adjusted for total population, and oral use of contraceptives (dichotomous) was additionally adjusted for females.

Discussion
Our years and ≥ 60 years old subgroups also had increased risk of VD inadequacy. After stratifying by gender, most of the above-mentioned effects persisted for both genders and were more pronounced in female participants. Our analyses indicated that there was somewhat age-and gender-difference for the effects of tobacco smoke exposure on VD levels. Our results also provided some evidence concerning impacts of tobacco smoke exposure on VD intoxication, which was rarely investigated in previous studies of the same content.
Our ndings for relationship between tobacco smoke exposure and serum VD concentrations were partly supported by previous epidemiological studies. A Norwegian study on 205 participants aged ≥ 29 years found that serum 25(OH)D levels were signi cantly lower in smokers than nonsmokers (Jorde 2005  higher prevalence of VD de ciency and inadequacy (Manavi 2015). Although these epidemiological studies were limited to adults, their ndings supported our results that tobacco smoke exposure, including active and passive smoking, was associated with increased risk of VD de ciency and inadequacy. It is noteworthy that the associations for smoke exposure with VD status were signi cant for participants with different races and BMI categories, indicating the adverse effects of exposure were stable.
The mechanism behind the disrupting effects of tobacco smoke on VD are unclear. On the ground of the foregoing experimental and epidemiological evidence, possible mechanisms for tobacco smoke exposure to interfere with VD were recently summarized as several highly likely pathways (Mousavi 2019). First of all, smoking could induce skin aging, and smoking-derived aging may disturb the cutaneous production of VD.
Second, dysfunctional VD-parathyroid hormones (PTH) axis due to tobacco smoke exposure could result in disruption of the VD metabolism. In addition, it appears that tobacco smoke is associated with dysregulation of enzymes genes related to the metabolism of VD. Another possible pathway is renal tubular dysfunction caused by tobacco smoke exposure. Heavy metals contained in tobacco may accumulate in kidneys, inhibiting VD activation through impairing kidney function. Besides, it is also hypothesized that tobacco smoke could depress intake of VD due to changed dietary taste. Although the exact explanation related to age difference is unknown, we assume that the observed protective effects of tobacco smoke exposure on VD levels in young people in our study might due to the very small numbers of exposed subjects. Nonetheless, further investigation is warranted to clearly ascertain mechanisms responsible for the reported smoking-VD associations as well as age-and gender-differences.
Our study has multiple strengths. First, our study used a nationally representative sample with a large sample size, which allowed for exploring age-and gender-difference in the associations between tobacco smoke exposure and VD levels as well as potential modifying effects of several important factors. Second, the study provided important evidence for long-term trends in tobacco smoke exposure over a 14-year period. Of note, smoking rates were consistently high over time, especially for active smoking among male adults and passive smoking among children. Third, our study was the rst to investigate effects of tobacco smoke exposure on VD intoxication, giving more clues on the disruptive role of tobacco smoke.
Our results, however, should be interpreted with caution due to following limitations. First, given the crosssectional nature of this study, no causal inference could be derived. Future evidence from prospective study design is warranted. Second, cotinine has a short half-life, and the measurement was based on a single spot serum sample. Therefore, the indicators detected could only represent a short-term level and the variation of individuals might be overlooked. Second, the lack of data on sun exposure such as season and latitude, and In conclusion, serum cotinine was signi cantly and inversely associated with 25(OH)D in adult participants.
Tobacco smoke exposure, including both active and passive smoking exposure, was associated with increased risk of VD de ciency in adults. Moreover, active smoking of adults was additionally related to increased risk of VD inadequacy. These associations showed somewhat gender difference, with consistent and stronger associations observed in female adults. In contrast, the effect of tobacco smoke exposure in children and adolescents aged 3-19 years on VD levels were mostly protective or non-signi cant. More researches are needed to verify our results.

Declarations
Ethics approval and consent to participate The NCHS Research Ethics Review Board (ERB) approval and documented consent was obtained from all participants.
Consent for publication Not applicable.
Availability of data and materials: The data underlying this article will be shared on reasonable request to the corresponding author. The datasets were derived from sources in the public domain: CDC. NCSH. National Health and Nutrition Examination Survey. https://www.cdc.gov/nchs/nhanes/index.htm.
Competing Interest: The authors declare no actual or potential con icts of interest.
Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Authors' contributions: Conception, methodology, writing, validation (Lei Yuan); Methodology, software, formal analysis, visualization (Jingyi Ni). All authors read and approval the nal manuscript.  Both cotinine and vitamin D were ln-transformed. Estimates were presented as coe cients and 95% con dence intervals (CIs) and were adjusted for age (continuous), BMI (categorical), ethnicity/race (categorical), PIR (continuous), NHANES cycle (categorical). Gender was also adjusted in the total population, oral contraceptive use was adjusted for females aged ≥12 years, and kidney health (categorical) was adjusted for participants aged ≥20 years.