The rate of absorption of Mo depends, inter alia, on its solubility. In contrast to MoO3, MoS2 particles are practically insoluble in water, hence their absorption from the lung tissue is expected to proceed at a very slow rate [21, 22]. Quantitative estimates of absorption following inhalation exposure to molybdenum in humans or animals were not identified [23]. Some evidence for absorption of molybdenum trioxide from the airways mucosa is available from inhalation studies on molybdenum trioxide conducted in rodents, i.e. in guinea pigs [24] well as rats and mice [25]. To our knowledge, our study is the only one available in the published literature which evaluated kinetics parameters in rats exposed via inhalatory route to MoS2 particles, hence any reliable comparisons to the published data cannot be made.

Available data on Mo kinetic parameters after exposure via other routes indicate its rather fast absorption, distribution and elimination, depending to a great extent on the chemical form of Mo. In the study conducted by Werner et al. [26] on volunteers, the elimination of Mo from the blood after a single intravenous injection of a trace quantity of Mo (ranging 300‒450 μg) occurred in the T1/2 range of 4‒70 min (half the time of the fast component of clearance) and in the T1/2 range of 3‒30 h (half the time of the slow component of clearance) in a two-compartment model. In the presented biokinetic model, the authors claimed that the clearance of plasma was much faster than the literature data. In addition, the authors showed that the volumes of distribution were significantly higher than the plasma volumes, but smaller than the calculated extracellular spaces. The authors further claimed that the faster Mo clearance from plasma might be explained by a quick uptake of Mo into tissues. This may indicate a very fast distribution of Mo in body fluids. The slight differences in results observed in our paper may be explained by both physiological inter-individual differences and also by the sampling schedule. Moreover, we also assume it may be related to the changing physiology of animals with maturation leading to lower demand for Mo in a growing organisms and eventually lower Mo plasma concentration. Unfortunately, we were not able to identify studies which could provide any support for such hypothesis (and as mentioned above no studies were found on relevant Mo blood kinetic parameters after inhalation exposure to particulate forms of Mo). It can be assumed that an unknown fraction of each dose administered after various time intervals was absorbed from the lungs (not known if in the form of particles or ions released after dissolution of the particles), causing comparable spikes in Mo concentration, but it was afterwards efficiently eliminated from the blood before the next dose. It is probable that within 2 weeks between administrations an equilibrium of the Mo distribution between the tissues and the blood has been established, which did not lead to increased Mo blood concentrations. As we calculated, the expected elimination half-life was T1/2 354 vs. 195 days for the higher dose (5 mg kg−1) of MoS2-MPs vs. MoS2-NPs, respectively, compared with an elimination half-life of T1/2 221 vs. 212 days for the lower dose (1.5 mg kg−1) of MoS2-MPs vs. MoS2-NPs, respectively. Based on these data, we hypothesize that, after repeated dosing, there is quite a rapid absorption of a certain amount (most probably very small) of the particles fraction administered (including ions after dissolution), while the remaining part of the particle fraction accumulates in the lungs. Moreover, For MoS2-MPs it could be seen that after 90 days the concentrations were higher than at the beginning of the experiment, so it was excreted more slowly. This is consistent with the observations of Kuraś et al. [27], as a lot of MoS2-MPs are observed in the lungs, the same MoS2-MPs absorption into the blood is faster, greater and excretion longer, as confirmed in Fig. 6.

Turnlund and Keyes [28] conducted a study on the clearance of Mo from the blood in men after administration of Mo, first intravenously (33 μg of 97Mo) and then orally (22 μg day−1 Mo). The administration of Mo increased both the natural intrinsic Mo in plasma and the total Mo in plasma during the first minutes (6.9 vs. 6.9 nmol L−1, respectively) up to 1 h (13.0 nmol L−1 vs. 17.1 nmol L−1, respectively), then it again decreased to near baseline after 24 h of uptake (5.7 nmol L−1 vs. 6.0 nmol L−1, respectively). Eventually, 48 h after infusion Mo concentration remained at a similar but only slightly lower level (5.1 mmol L−1 vs. 5.3 mmol L−1, respectively). After 72 days, Mo concentration remained unchanged (5.8 ± 2.5 nmol L−1). These findings on urinary excretion are in agreement with the data obtained by Werner et al. (2000). The authors of the study suggest that the introduced Mo disturbed the overall Mo metabolism at the beginning of the experiment. It resulted in an increased level of Mo after exposure, combined with the physiological level of natural Mo. More specifically, Mo could have been absorbed by the tissues that released the pool of bioavailable intrinsic Mo in the body increasing its concentration in the blood. Further exposure to low dietary Mo may have resulted in physiological adaptation [28]. Another study concerning compartmental modeling to explain the alteration in Mo distribution and excretion with the urine showed a positive correlation in the studied men, where increased Mo intake was associated with both increased Mo absorption and urinary excretion. The fraction deposited in tissues was inversely correlated [29]. It is known that Mo is mainly excreted in the urine and it is a key pathway for modulating exposure to Mo in the body. Molybdenum from feces is eliminated in lower amounts. In humans, it is up to 17‒80% of the total absorbed Mo dose [30, 31], but Giussani et al. [32] and Novotny and Turnlund [29] reported that this excretion was on the level of 75–90% of the absorbed Mo dose. Urinary Mo excretion, according to the results obtained by Bell et al. [33] after oral administration to rats, showed that 90% of the dose was eliminated by the kidneys. The lack of multiple urine collection from the freely moving rats may be considered a limitation of this article. This was not included in the study implementation schedule because attention was focused on the intratracheal instillation exposure and on following the Mo metabolism and key pathways of its regulation connected with blood kinetics and tissue distribution.

The latest research has revealed that MoS2 has the ability to cause inflammatory reactions [31, 34]. It was shown that MoS2-MPs as well as MoS2-NPs deposited in the lung tissue of the rats after intratracheal instillation may cause inflammatory reactions, although a stronger response was observed for MoS2-MPs. The authors observed inflammation in the respiratory system in the rats after a single administration. The difference in the inflammatory response was statistically significant for both doses (1.5 and 5 mg MoS2 kg−1 b.w.) 7 days after the autopsy for MoS2-MPs compared to control (PVP) rats [15]. Moreover, the authors showed interstitial inflammation at a higher dose, both 24 h after the autopsy (for both forms) and 7 days after the autopsy for MoS2-MPs. This data is confirmed by the results presented in our paper. STEM with EDS unambiguously revealed multiple alveolar macrophages loaded with plate-shaped Mo-MPs as well as agglomerates of Mo-NPs. The characteristically expanded lysosomes in these macrophages containing similar clusters of particles were observed in the cytoplasm of macrophages. The authors also showed the presence of NPs in epithelial cells, which may suggest that the process of internalization indicates the possibility of NPs penetration through the epithelium and systemic circulation extended clearance [15]. Chng et al. [35] noticed that disk-shaped particles were conducive to proinflammatory reactions in the respiratory system. Moreover, the histopathological assessment after chronic inhalation of 6.6 mg MoO3 mg/m3 in mice revealed significantly greater instances of adenoma or carcinoma of alveolar/bronchiolar in the exposed groups in comparison to control ones [36]. Furthermore, in the same study the authors pointed to marginally greater incidents of lung tumor in male rats. The initial histopathological lung damages were observed already at 10 mg/m3. In another study, Huber and Cerreta [37] reported an increase in the neutrophils and multinucleated macrophages in BAL fluid in hamsters after one day of inhalation of 5 mg Mo/m3, and lymphocytes after 7 days of exposure. The increase in neutrophils in BAL fluid was also observed in mice after inhalation of 90 mg Mo/m3 [16]. What is more, the tidal volume was already decreased after the lowest exposure level (8 mg MoS2/m3). Another study conducted by Peña et al. [17] also confirmed a lung inflammation caused by MoS2 nanosheets after a single inhalation in mice. Inflammatory cytokines and extracellular vesicles as well as immune cells detected in BAL fluid effected on inflammatory status.

Another study assessed the toxicity of Mo-NPs on rat BRL3A, i.e., rat liver cells, after 24-h exposure [38]. The authors of this study observed a significant increase in the lactate dehydrogenase enzyme release at the Mo-NPs concentration of 250 μg m L−1—much higher than in the study conducted by Braydich-Stolle et al. [39]. Also, an increase in mitochondrial activity reduction occurred at a much higher concentration—250 μg m L−1 [38]. Mo supplementation significantly increased the activity of xanthine dehydrogenase/xanthine oxidase, sulfite oxidase and superoxide dismutase in the liver [40]. This is also confirmed by a study conducted by Yang and Yang [41]. The authors investigated an effect of Mo supplementation (0.1 mg Mo L−1) of rats on the concentration of hepatic Mo, which was increased significantly compared to controls. Thus, it is very likely that the increased concentration of Mo in this study, observed after 7 administrations, caused disturbances in the metabolism of liver enzymes due to tissue retention.

Moreover, Mo is an essential trace element, which, as an enzyme component, supports iron metabolism and thus contributes to hematopoesis. Accumulation of Mo in tissues can cause the risk of anemia [42, 43]. In our study, we observed deviations in basic hematological parameters in exposed animals. Similar to Sobańska et al. [15] study. Therefore, lower Mo concentrations in blood after exposure are associated with hematological changes and damage to the vascular system during material collection, that directly affects hematological parameters (decrease in red blood cells, lower Mo concentrations). Kusum et al. [44] obtained similar results. According to authors, oral exposure to Mo in goats may altered haematological profile, because it causes a state of secondary copper deficiency. As a consequence, the study revealed significant reduce in mean hemoglobin, packed cell volume, total leukocyte as well as erythrocyte count. The mean of corpuscular hemoglobin concentration was also significantly decreased. Lyubimov et al. [45] confirmed a decrease in erythrocyte count as well as hematocrit in rats after gavage administered by 4.4 mg Mo/kg/day. Moreover, in the study Asadi et al. [46] the number of white blood cells increased with increasing levels in Mo NP dosage, after intraperitoneal injections in rats. NPs cause inflammation due to disorders in the lymphatic system.

In the described study, rats were exposed by intratracheal administration to nano- and micro-metric forms of Mo. It can be concluded that, after such exposure, MoS2-NPs as well as MoS2-MPs were mostly retained in the lung tissues. Distribution of the administered molybdenum disulfide particles was also observed in extrapulmonary tissues. Repeated exposure resulted in a significant accumulation of particles in both lungs and other tissues, with the following order of concentration: liver > spleen > brain. The distribution exponent was the fastest for the lower nanoparticle dose at T1/2 8.8 days. The calculated elimination half-life was also faster for the nano-forms of Mo in comparison to the micro-forms, regardless of the dose.

The present results provide a solid basis for further research on the fate of nanoparticles in the body. Additional studies, such as information on the extent of oral exposure after inhalation exposure, are necessary to clarify the routes of exposure. In addition, this is the first study in which 3 techniques were used to complement each other for the evaluation of the effects of intratracheal instillation of MoS2-MPs and MoS2-NPs on tissue distribution in rats. The LA-ICP-MS technique was proposed as a complementary tool for ICP-OES and ICP-MS, for the identification as well as bioimaging of different sizes of Mo particles in rat tissue. The impact of the particle size and form was investigated, which may be an important tool in further internal biokinetics studies. Intratracheal exposure to Mo particles showed their retention and deposition, mainly in the lung tissue, in the form of MoS2-MPs, and to a lower extent in the liver and spleen, but mainly in the form of MoS2-NPs. Taking into consideration the complex nature of factors determining the toxicity of NPs and MPs, biological as well as toxicological studies should be carried out multidirectionally.