The novel finding of this study was a highly significant increase in lung tumor multiplicity in mice promoted with SS welding PM, which was consistent and significant across all five individual lung regions. This response was observed 30 weeks after MCA-initiation. Also of note, there were more malignant lesion types in the MCA/high dose welding PM group which suggests that not only is the rate of tumor formation being increased by welding PM, but the progression to malignancy appears to be affected with higher doses of particulate. This study is the first to link enhanced lung tumor formation and welding fume exposure in vivo and provide animal evidence to support epidemiological findings.
A 25% to 40% increased risk of lung cancer has been associated with the welding occupation [14–16]. Indeed, the proportionate mortality ratio for welders for lung cancer is 1.2 . Even though some evidence exists to the contrary, epidemiological studies generally support an increased risk, but they are limited in number; animal studies are scarce . Because welders work under widely diverse conditions and co-exposures such as silica, smoking, and asbestos may be involved, cumulative exposure data and a complete occupational history may not always be available [15, 18, 19]. Therefore, controlled animal studies to elucidate the underlying factors of welding fume-related lung carcinogenesis are long overdue.
Previously, we assessed the ability of different types of welding PM to act as a complete lung carcinogen in lung tumor susceptible A/J mice. Efforts of those studies were ultimately negative but hinted at a potential weak carcinogenic effect of SS welding PM, as a borderline significant (p = 0.057) increase in grossly observed lung tumor incidence (i.e., presence or absence of tumors) was found [10, 11, 20, 21]. In addition, histopathology at 78 weeks after exposure revealed presence of SS welding PM which was associated with a mild, but significant, chronic inflammatory cell influx in the lung tissue. Of note, these effects were not observed following exposure to mild steel (MS) welding PM composed largely of iron oxide . Also, to complement those studies, the lung toxicity and gene expression profiles in the tumor susceptible A/J and resistant C57BL/6J (B6) mouse were compared following pharyngeal aspiration of GMA welding PM [11, 22]. Interestingly, a significantly greater magnitude of overt lung toxicity (polymorphonuclear leukocyte influx, lung cytotoxicity and permeability) and an attenuated resolution of the inflammatory response to different types of welding PM were found in the A/J versus the B6 mouse strain. Results from the microarray analysis confirmed those aforementioned responses and revealed a greater lung transcriptional gene activation as well as a prolonged dysregulation of immunomodulatory genes after welding PM exposure in the A/J versus the B6 mouse . In all cases, the lung toxicity and transcriptional effects were greater with the carcinogenic metal-containing SS welding fume when directly compared to a MS welding fume. Therefore, historical data in our laboratory suggested that fumes containing Cr and Ni were the most toxic, persisted in the lung longer as compared to other types, and were possibly tumorigenic in vivo.
The A/J mouse lung tumor bioassay is well-characterized, has good inter- and intra-laboratory reproducibility, and has been widely used for testing hundreds of potential lung carcinogens, such as tobacco smoke and polycyclic hydrocarbons [23–25]. It also continues to be useful for evaluation of chemointerventive agents of lung neoplasia [24, 26, 27]. This strain is susceptible to both chemically-induced and spontaneous lung adenomas compared to the resistant B6 mouse . Morphological, molecular, and histological features of the lung tumors that arise in these mice resemble human adenocarcinomas; therefore, findings in this model have direct human relevance . Indeed, tumor susceptibility in the A/J strain has been associated with a polymorphism in intron 2 of Kras and this finding is pertinent to human lung adenocarcinoma development because ~35% of these human tumor types contain Kras oncogenes [29, 30].
In humans, chronic lung inflammatory conditions such as asthma and chronic obstructive pulmonary disease are associated with increased risk of lung cancer and epidemiology suggests ~25% of human cancers are attributed to chronic inflammation [31–33]. Microenvironments of chronic lung inflammation, largely dominated by macrophages and other leukocytes, create a milieu rich in reactive oxygen species and cytokines that may promote tumorigenesis [34–36]. Indeed, in the mouse model, quantitative trait loci (QTL) that control genetic susceptibility to lung inflammation colocalize with tumor susceptibility QTL . Two-stage carcinogenesis in lung tissue was first reported by Witschi et al. when repeated IP injections of butylated hydroxytoluene (BHT), a synthetic food additive and antioxidant, increased lung adenoma multiplicity in both Swiss-Webster and A/J mice initiated with a single dose of the potent carcinogen urethane 9–15 weeks prior . The model of BHT tumor promotion continues to provide mechanistic insight into the critical role that inflammation has in lung tumor initiation and promotion [35, 39, 40]. More recently, this two-stage model was used to demonstrate the in vivo promoter activity of vanadium pentoxide (V2O5), a component of environmental and occupational PM, in A/J, BALB/cJ, and C57BL/6 J mice initiated with MCA 20 weeks prior . In agreement with our earlier findings that strain-dependent (A/J > B6) lung responses were evident after welding PM exposure, Rondini et al. found that V2O5-mediated lung inflammation and subsequent tumor multiplicity also showed strain dependency (A/J > BALB/cJ > B6) [11, 12, 22]. In this study, the observed inflammatory cell infiltration and highly significant increased tumor multiplicity after MCA/GMA-SS welding PM exposure, combined with our previous evidence of chronic inflammation due to SS particle persistence in the lung, further supports the role of inflammation in the promotion of lung tumors in A/J mice.
GMA-SS welding PM is poorly soluble and contains toxic metals, namely Cr(VI), which is carcinogenic, especially in the particulate form . In vitro, this fume has also been shown to cause greater DNA damage, lipid peroxidation, and radical generation compared to MS fume . Increased DNA damage has also been reported in blood leukocytes from welders exposed to Cr and Ni fumes [42, 43]. Once inhaled, Cr(VI) particles are retained primarily by the lung and tend to accumulate near major bifurcations, where they may persist for as long as twenty years [44, 45]. A possible mechanism for Cr(VI) carcinogenicity involves the slow release over time of chromate ions from particulate compounds adhered to the cell surface. These ions may escape extracellular reduction by ascorbate, which then allows for uptake by lung epithelial cells causing tumor formation . Indeed, a concentration-dependent induction of aneuploidy has been shown in normal human bronchial fibroblast and immortalized human bronchial epithelial cells exposed to particulate chromate [47, 48]. In addition, Cr(VI) may act synergistically with Ni, present in lower amounts in this fume, as suggested by co-mutagenicity studies . In rats and mice, GMA-SS fume exhibited a slower lung clearance timeline compared to a more soluble manual metal arc-SS (MMA-SS) and GMA-MS fume [11, 50]. Thus, slower lung clearance, together with the greater lung toxicity profile of this fume in vivo, may significantly contribute to its increased tumor promoter activity.
No threshold limit value-time weighted average (TLV-TWA) exists for welding fume. The previous TLV-TWA of 5 mg/m3 for welding fume was retracted in 2004 by the American Conference of Governmental Industrial Hygienists . In this study, we used welding PM exposures equivalent to 1.84 and 3.67 years of work exposures at 5 mg/m3 and Cr(VI) exposures of 5.4 and 10.8 years at 5 μg/m3 in a human for the low and high doses, respectively. Previously published reports have indicated that airborne concentrations of Cr(VI) in industries using SS welding can be 50–400 μg/m3
. Welders oftentimes work in confined spaces which can increase the total fume exposure to > 20 mg/m3
. Because freshly generated welding fume induces greater lung inflammation than “aged” fume, such as that used in this study, and one-third of the dose by inhalation results in about 2 to 3 times the pulmonary toxicity, the exposures used herein are reasonable [11, 21, 54]. However, the bolus delivery of the particles is an obvious limitation in this study, even though the doses were repeated over a five- week time frame (1 exposure/week). As such, welding fume by inhalation is 6 to 9 times more potent than by pharyngeal aspiration . The mechanisms of increased toxicity by inhalation are likely related to the free radical generation of freshly generated welding fume compared to “aged” fumes that are collected onto filters then used in instillation studies [41, 54]. Given the role of inflammation in tumor promotion described above, the combined interpretation of our previous studies strongly suggests that a significantly lower mass deposition by inhalation would have similar results as those in this study.