Endotoxin, a component of the cell wall in Gram-negative bacteria, causes profound inflammatory responses in humans. Endotoxin is ubiquitous in the environment and is present at high concentrations in urban schools,¹ homes burning biomass fuel,² tobacco smoke,³ and a number of occupational settings, particularly cotton textile work.⁴ Endotoxin is an unusual exposure in that it is protective against childhood development of asthma⁵ and adult development of lung cancer,⁶ but harmful in terms of asthma development and morbidity with later life exposures,⁷ malignancies,⁸ cardiovascular disease,⁹ heart failure,¹⁰ and hepatic disease.¹¹

There remains debate about whether chronic exposure to the endotoxin component of organic dust ultimately leads to abnormal lung function¹² in a healthy working population with often better than predicted lung function. The effects of cotton dust have been best studied in the context of respiratory effects, where an asthma-like syndrome called byssinosis was first described,¹³ with later studies demonstrating an accelerated decline in lung function.¹⁴ While early longitudinal studies in cotton textile workers suggested that men had a greater loss of lung function compared to women,¹⁵¹⁶ many of these studies have been limited by the healthy worker survivor effect,¹⁷¹⁸ incomplete exposure assessment, loss to follow-up exceeding 50% of the original cohort, relatively short duration of follow-up, and most importantly, inadequate control of smoking as a confounder. Thus it remains unclear, for example, whether any observed adverse pulmonary effects or differential gender effects were due to residual confounding from smoking. Additionally, there did not seem to be a clear biologic basis to suspect that gender differences would exist in the context of cotton dust exposure. It must be noted that many of these longitudinal studies focused on dust and not endotoxin as the exposure of interest. As cotton from various regions or growing conditions can contain different amounts of endotoxin, gravimetric measurements of dust exposure cannot always serve as a proxy for endotoxin exposure.¹⁹

In recent years, however, studies performed in both animal models and experimental human studies show an augmented inflammatory response to endotoxin in males compared to females.^20-22 Most studies of endotoxin exposure are performed in the acute setting, and it remains unknown whether these gender differences persist in the chronic setting where repeated high level exposure occurs.

The Shanghai Textile Worker Study provides a unique opportunity to study the associations between gender, occupational endotoxin exposure, and time to development of impaired lung function or death. In this cohort study, cotton and silk textile workers have been prospectively followed with repeated measurement of occupational endotoxin, smoking habits, and lung function every five years, with a 74% follow-up rate at 30 years. Cotton textile work is associated with extremely high levels of airborne endotoxin because as cotton plants grow and the boll opens, they become increasingly colonized by gram negative bacteria, leading to one of the highest endotoxin levels measured in any setting.⁴ In contrast, silkworm cocoons are boiled to prevent the larvae from emerging and destroying the silk fibers as well as to facilitate unraveling, and so the boiled cocoons have minimal bacterial contamination and minimal endotoxin (endotoxin is water soluble). The raw material provided to silk textile mills in this study was raw spooled silk, while the raw material provided to the cotton textile mills were ginned cotton bales heavily contaminated with endotoxin. Thus this natural experiment allowed us to assess the time to development of impaired lung function and death in relation to occupational endotoxin exposure in the Shanghai Textile Worker Study, and furthermore to determine whether the effects of exposure differ by gender.

Of the 1021 textile workers who met our eligibility criteria in the 3 mills, 919 (90%) participated in the 1981 baseline survey. There were 472 silk and 447 cotton textile workers. The median follow-up time was 30 years with no significant differences in average follow-up time between cotton and silk workers (see Figure 1 for schema detailing study design, events, and follow-up). Table 1 shows the baseline demographic and lung function characteristics of the study population in 1981. Cotton textile workers were more likely to be smokers and to have been employed for over 5 years. Males were more likely to be older, smoke, and among cotton textile workers, to have been exposed to higher cumulative levels of endotoxin.

128 deaths and 164 diagnoses of impaired lung function were ascertained between October 1981 and August 2011. Causes of death were related to neoplasms (n=60, with 11 due to lung cancer), diseases of the central nervous system (n=35), circulatory system (n=19, of whom 4 had chronic pulmonary heart disease or cor pulmonale), respiratory system (n=6), digestive system (n=2), genitourinary system (n=3), injury, poisoning, and certain other consequences of external causes (n=1), other external causes (n=1), and abnormal clinical and laboratory findings not otherwise classified(n=1). The diagnosis of impaired lung function was made more frequently in men vs. women (106 vs. 58, men vs. women), and deaths occurred more frequently in men (89 vs. 39, men vs. women).

Kaplan-Meier curves for the unadjusted risk in cotton vs. silk workers of the composite endpoint of impaired lung function and death (Figure 2), and for the unadjusted risk of all-cause mortality (Figure 3) showed significantly worse outcomes for cotton vs. silk workers.

In multivariate models, cotton vs. silk worker status was associated with a 47% increase in the risk of a composite outcome of impaired lung function or death (HR 1.47, 95% CI 1.09 – 1.97), Table 2. The association differed by gender (cotton by male interaction p-value=0.04), and was elevated only in male textile workers (HR 1.84, 95% CI 1.27 – 2.67 in males vs. HR 1.00, 95% CI 0.62 – 1.61 in females). When exposure was modeled as cumulative occupational endotoxin exposure, it was likewise associated with an increased risk of impaired lung function and death, with each 10,000 EU/m³-yrs increase in cumulative endotoxin exposure associated with a 4% increase in risk (HR 1.04 95% CI 1.01 – 1.07). Risk was elevated in males (HR 1.05, 95% CI 1.01 – 1.08) but not females (HR 1.00, 95% CI 0.91 – 1.10).

Cotton vs. silk worker status had a similar magnitude of risk for all-cause mortality as for the composite outcome, although this association was not statistically significant in the overall cohort (HR 1.36 95% CI 0.93 – 1.99). There was a higher risk of death in male cotton compared to male silk workers (HR 1.63, 95% CI 1.04 – 2.57); this was not observed in female cotton vs. female silk workers (HR 0.92, 95% CI 0.47– 1.77). There was no statistically significant increase in death associated with cumulative endotoxin exposure in the overall cohort (HR 1.04, 95% CI 0.99 – 1.09) although the magnitude of risk again was again elevated in males (HR 1.04, 95% CI 1.00 – 1.08) but not in females (HR 1.00, 95% CI 0.89-1.14).

We also report the cause-specific hazard associated with exposure for the development of impaired lung function in order to verify that exposure is associated with both components of the composite outcome in the same direction. This analysis has to be interpreted carefully in light of the issues of semi-competing risks and the known association between low FEV₁ and death from both pulmonary and non-pulmonary causes. We observed a similar magnitude of risk for development of impaired lung function associated with exposure. Given the low event rates (note the 47 subjects who already had impaired lung function in 1981 were effectively excluded from this analysis), the point estimates for the gender specific analyses are less precise and therefore more difficult to interpret.

While we adjusted for the confounding effects of smoking, given that 94.8% of the female but only 22.9% of the male cotton textile workers remained nonsmokers throughout the duration of the study, we performed a sensitivity analysis to verify that the gender differential in the effects of occupational endotoxin exposure on health was not due to residual confounding from smoking. In the 583 textile workers (313 silk, 270 cotton) who remained nonsmokers throughout the duration of the study, we observed the same patterns of risk as observed in the analysis on the entire cohort (Supplemental Table 1). As expected, confidence intervals were wider due to lower event rates in this non-smoking subgroup.

In the largest observational study of textile workers to date with the longest duration of sequential follow-up, we found that occupational endotoxin exposure is associated with an increased risk of either impaired lung function or death; a similar increase in risk is seen for all-cause mortality alone. The observed risk was elevated only in male and not in female cotton textile workers.

We identified a striking gender effect on the impact of long term occupational endotoxin exposure on impaired lung function and death; this is the first study to clearly demonstrate that risk associated with occupational endotoxin exposure has significant gender differences. While other longitudinal studies of cotton workers have found a trend towards a greater annual decline in lung function in men as compared to women, they have been limited by small study size, lack of control for confounders, no exposure assessment for endotoxin, and high loss to follow-up. ¹⁶¹⁵³⁶

This is also the first study to evaluate the effects of occupational endotoxin exposure beyond pulmonary health with the use of a composite outcome. This was motivated primarily from the biological standpoint to address the known pleiotropic health effects of endotoxin although it also addressed the issue of semi-competing risks with increased statistical power.²⁹ While the respiratory health effects of endotoxin are the best studied, the health effects of endotoxin clearly extend beyond the respiratory system.^5-11 When evaluating the components of the composite outcome in a sensitivity analysis, we find the same consistent relationship; cotton dust exposure is associated with an increased risk of both low lung function and death. If the relationship between exposure and each outcome was not consistent, then a loss of power would result when combining these two outcomes, and not a gain of power as we have observed.

Gender effects on the risk associated with occupational endotoxin exposure could be explained by a number of different factors. Occupational endotoxin exposure was higher in male compared to female cotton textile workers; men were more likely than women to work in highly exposed areas such as opening, carding, and blowing, which exposed them to higher levels of endotoxin (although no additional occupational exposures besides cotton dust was present in these work areas). Thus a threshold effect of occupational endotoxin on health could be postulated. In any observational study there is the potential for uncontrolled confounding, and of greatest concern in this study was that almost none of the female workers were smokers and thus gender and smoking was highly correlated. When smokers were excluded from the analysis, however, we observed the same differential effect of occupational endotoxin exposure on both endpoints, suggesting that the interaction between gender and endotoxin exposure is not due to residual confounding from smoking. Alternatively, a true gender effect could be present. Human studies have demonstrated that women have an attenuated physiologic response to low dose endotoxin injection²¹ and decreased production of inflammatory cytokines such as TNF-α³⁷ when compared to men. Estradiol appears to play a role in modulating the response to endotoxin in in vitro studies,²² and animal studies have demonstrated that intratracheal endotoxin administration leads to exaggerated inflammatory responses in male compared to female mice; this effect is attenuated with gonadectomy in male mice and similarly accentuated in females with the administration of testosterone.²⁰ Thus a biologic explanation for the differential response to occupational endotoxin exposure by gender is clearly present, and would identify men as a vulnerable subgroup in these highly endotoxin-exposed settings.

Only one other study³⁸ has compared mortality rates in cotton vs. silk textile workers although this study focused on COPD-related mortality and reported a higher risk in silk compared to cotton textile workers. This registry study based on the Shanghai Textile Industry Bureau involved 267,400 female textile workers with mortality data obtained from 1989 to 2000. Chronic obstructive pulmonary disease (COPD)-related mortality as reported on death certificates was higher in cotton workers compared to the general textile population but unexpectedly highest in silk workers. Curiously, COPD deaths were observed only among silk workers who did not smoke; no COPD deaths were recorded in smoking silk workers. One explanation could be that sicker silk workers would be less likely to smoke than healthy silk workers, i.e. a “healthy smoker effect” that has been previously described.³⁹ Given the lack of exposure assessment and detailed medical histories in these subjects as well as low event rates,¹⁸⁴⁰it is difficult to determine whether silk dust actually represents a toxic exposure or whether these results are related to a differential healthy worker survivor effect that depends on the toxicity of the exposure where cotton dust represents the more toxic exposure.⁴⁰⁴¹ It is likely that the general cotton textile population is healthier than with general silk textile population. This is consistent with prior correspondence between one of the authors (DC) and physicians working for the Shanghai Textile Bureau indicating that by policy, candidate workers with any prior respiratory disease are automatically excluded from cotton textile work. Our study population was drawn from a subset of this registry based study – we explicitly excluded silk workers with a prior history of active tuberculosis or asthma, thus selecting for healthy silk workers as a comparison group. We observed decreased, not increased overall mortality when comparing previously healthy silk with previously healthy cotton textile workers, although our sample size did not allow us to specifically address COPD-specific mortality.

The major strengths of this study are its prospective design with working matched controls, duration and frequency of follow-up, serial assessment of endotoxin exposure, smoking habits, spirometry, and high participation rate even at 30 years after the baseline survey. Few studies have repeated spirometry performed in subjects without overt respiratory disease. Additionally, we have carefully accounted for the presence of left truncation, left censoring, and semi-competing risks present in this study. We have also performed sensitivity analyses to demonstrate the consistency of our findings.

We acknowledge that our study has several limitations. First, we estimated occupational endotoxin exposure from job histories and area samplers for dust and endotoxin rather than from personal samplers. Additionally, exposure assessment was not performed prior to 1981 and the assumption was that endotoxin and dust levels did not change significantly in the cotton textile mills. No significant changes to the infrastructure and ventilation system of the mills were made since the 1950s although if the origin of the baled cotton used in the mills changed significantly in the decades prior to 1981,⁴² this assumption may be less valid. These above factors may contribute to exposure misclassification. Personal samplers are still not available for measurement of cotton dust exposure, and by study design we could not have obtained measurements of endotoxin and dust levels prior to 1981. However, such exposure misclassification would be expected to be non-differential and bias results towards the null; in our study we still observed significant results despite these limitations. Second, our cohort was not a newly-hired cohort but rather was composed of prevalent hires raising the concern for a healthy worker survivor effect.¹⁸⁴⁰ We adjusted for the number of years worked as well as left truncation in order to address this issue; An analysis using Cox proportional hazards models stratified by the number of years worked at study inception to account for the healthy worker survivor effect also showed no significant differences in our results. If present, the healthy worker survivor effect would again be expected to bias our results towards the null. Third, it is possible that other exposures correlated with endotoxin exposure may play a causative role in disease development. Animal studies have supported the role of endotoxin in causing respiratory pathology⁴³ and in particular the etiologic role of endotoxin in cotton dust.⁴⁴ Other agents such as β-glucan, a fungal cell wall component, may also play a role,⁴⁵ but evidence to date suggests that it is the endotoxin component of cotton dust that is the major contributor to respiratory disease. Fourth, while a 74% follow-up rate in a 30 year longitudinal study is unusually high, one may argue that those lost to follow-up may not be representative of those that continued to participate in the study. Since the primary research question relates to the effect of cotton vs. silk dust on the outcomes, if the loss to follow-up was different between cotton vs. silk workers, and if this was not adjusted for in our analysis, then there is the potential for biased estimates of the effect of cotton dust on low lung function and death. Survival analysis is particularly suited to dealing with incomplete (censored) time to event data and one reason why we chose this method of analysis. The censoring is allowed to be dependent on the exposures. In our case, since we always include the exposure in our models as it is precisely our primary covariate of interest (whether as cotton vs. silk worker status or as measured endotoxin exposure), we still get valid estimates of the effect of cotton dust or endotoxin exposure on the time to development of low lung function or death even if follow-up rates were to differ between cotton vs. silk workers.

The exact nature of the observed impaired lung function associated with occupational endotoxin exposure is unclear from this study. Prior studies have shown an association between endotoxin containing organic dust and the development of obstructive lung disease,¹⁴⁴⁶ although it is unclear whether the etiology of the observed airflow obstruction is due to small airways disease or emphysema. Post-bronchodilator spirometry and lung volumes were not obtained in this study, and thus we are currently unable to specifically address this issue. A study using high resolution computed tomography to answer this question is ongoing in this cohort.

In summary, occupational endotoxin exposure is associated with an increased risk of impaired lung function and all-cause mortality in Chinese textile workers. The observed risk was seen only in male and not female cotton textile workers, even after careful adjustment for the effects of smoking. High levels of endotoxin have been detected in cigarette smoke, homes burning biomass fuel, and various occupational settings such as agricultural work sewage treatment plants,^2-4 suggesting that our results have broader implications beyond the textile industry. If men are more vulnerable to the health effects of endotoxin than women, then they may represent a subgroup that will benefit from targeted interventions such as pre-emptive screening for sub-clinical disease, workplace measures to lower endotoxin exposure, and lifestyle modifications such as smoking cessation to reduce the risk of adverse health effects.