Technical Report 122

Understanding the mechanisms of the unique neoplastic rat lung responses to particle overload

Warheit et al (1997) performed a study that provides a mechanistic explanation for the responses observed in the pivotal rat oncogenicity study (Lee et al, 1985). The study demonstrated that the lungs of particle-overload exposed rats are characterised by impaired pulmonary clearance, sustained pulmonary inflammation, cellular hypertrophy and hyperplasia; and that these effects, following continuous exposure at 250 mg/m³ (for two years), likely could result in the development of overload-related pulmonary tumours.

The study with pigment-grade TiO2 in rats used exposure concentrations similar to those in the Lee et al, study (1985) to detail the characteristics of ‘lung overload’ in this species, along with an assessment of the rat's ability to recover from this challenge. Male rats were exposed to TiO2 particles 6 hours, 5 days a week for 4 weeks at concentrations of 5, 50, and 250 mg/m³ and evaluated at selected intervals through 6 months post-exposure. Exposure to high dust concentrations produced pulmonary inflammation, proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. Rats exposed to 250 mg/m³ TiO2 had lung burdens of 1600 mg/g of fixed lung tissue or 12 mg/lung. TiO2 particles produced sustained pulmonary inflammatory responses in animals exposed to 250 mg/m³, corresponding to substantial numbers of neutrophils recruited to alveolar regions. Rats exposed to 50 mg/m³ TiO2 had small, sustained inflammatory responses. Rats exposed to 250 mg/m³ demonstrated diminished lung clearance after 1 week through 1 month post-exposure. Mono-exponential clearance modelling indicated that TiO2 particles were cleared with half-times of approximately 68, 110, and 330 days for the 5, 50, and 250 mg/m³ test groups, respectively. Lymph node burdens of rats exposed to 250 mg/m³ TiO2 demonstrated TiO2 particles had translocated to tracheobronchial lymph nodes. In vitro phagocytosis studies demonstrated that alveolar macrophages exposed to 250 mg/m³ TiO2 were impaired in their phagocytic responses. At high concentrations (50 to 250 mg/m³) of TiO2, cellular hypertrophy and hyperplasia were evident at alveolar wall and duct bifurcations that were adjacent to the macrophage.

Despite a comparable behaviour of rats and humans to accumulate PSP within similar lung compartments, morphometric analysis by Nikula et al (2011) have shown that the relative amounts of intraluminal and interstitial particle load differ markedly between rats and humans with particles being found predominantly in the interstitium in man and intraluminarly in rats. Additionally, quantitative effect response analysis to PSP exposures revealed differences at cellular levels between rats and humans. Especially the occurrence of acute intra-alveolar inflammatory responses, alveolar epithelial hyperplasia and alveolar lipoproteinosis were all significantly more pronounced in rats compared to humans exposed to the same particles (Green et al, 2007). According to the authors, these differences may also account for the species differences seen in the long-term responses to high PSP exposures.

In evaluating mechanistic differences in the response of rats vs. mice or hamsters to inhaled carbon black particles, Carter and coworkers (2006) assessed the levels of several key pro- and anti-inflammatory mediators in the lungs of exposed animals. These investigators postulated that the unique response of the rat may relate to an inability to generate sufficient anti-inflammatory mediators in the face of continuing pulmonary particulate overload exposures. Thus, the aim of the study was to study pro-inflammatory and anti-inflammatory mechanisms underlying species specificity in carbon black-induced lung inflammation. Accordingly, rats, mice, and hamsters were exposed to carbon black particulates at 3 concentrations for 13 weeks as described in the Elder et al, 2005 study, at 3 concentrations (1, 7 and 50 mg/m3) for 13 weeks. Bronchoalveolar lavage along with reactive oxygen and nitrogen endpoints, and cytokine levels were measured. Ex vivo mutational analysis of inflammatory cells was measured by co-incubating with lung epithelial cells. In addition, lung tissue was evaluated for gene expression of various anti-inflammatory mediators. The investigators reported a dose- and time-course related effect with all the parameters.

Rats demonstrated greater propensity for generating a pro-inflammatory response, whereas mice and hamsters demonstrated an increased anti-inflammatory response. These incipient findings suggest a potential mechanism for delineating the differences in rat responses to particle overload-induced lung inflammation when compared to mice or hamster responses.