Blood biochemistry deals with the processes that occur within living cells and between cells which in turn relates greatly to the prediction of tissues, organs, and organism structure and function [39]. Oxidative stress and biochemical processes are closely related to several abiotic and biotic factors; among them, environmental insults are considered key players [40]. The literature already includes several reports of adverse effects of different pollutants on metabolic profiles of aquatic and terrestrial organisms; however, the effects of nano-scale plastics have been poorly elucidated [11, 40, 41, 42]. Even though microplastics (either virgin or with sorbed chemical pollutants) are known to induce various apical endpoints, toxicological investigations rarely use cellular biomarkers to test metabolic defects of nano-sized plastics, particularly in rodent species usually used for toxicity studies on which to base risk assessment for humans. The present study reports the first appraisal of toxicity and related biomarker responses induced by nano-scale plastics in rats and provides new insights into the possible mechanism for toxicity of nano-sized plastic particulates in humans.
Several macromolecules in the body including proteins, DNA and lipids are susceptible to the oxidative damage caused by these constituents [43]. The literature already includes several reports of ROS generation and the subsequent toxic effects such as genetic, mitochondrial and DNA damages in toxicity studies with engineered metal (oxide) nanoparticles [44, 45]. A number of studies have also, in agreement with our findings, reported the induction of oxidative stress caused by exposure to small pieces of plastics [46].
CAT plays a fundamental role in decomposition and reduction of H2O2 to water and oxygen by using either iron or manganese as a cofactor. CAT is responsible to directly scavenge reactive oxygen species. Although the difference was marginally significant (p = 0.07), it was possible to draw a meaningful conclusion relating to the impact of nanoplastics exposure on CAT activity pointing toward the large effect size (𝜂2 = 0.72) obtained. However, with a small sample size, caution must be applied as the results of CAT analysis are unreliable based on the confidence interval results, demonstrating that the study needs to be replicated with a larger sample size or meta-analysis.
The literature is somewhat contradictory with respect to the induction of oxidative stress and the antioxidant defense during exposure to microscopic plastic particles. Some studies have reported altered oxidative stress biomarkers concentrations, for example, increased activity of CAT following microplastics exposure in clam Scrobicularia plana [47], scleractinian coral Pocillopora damicornis [48] and African catfish (Clarias gariepinus) [49]. This same general trend was reported by Revel et al., 2019 [50], who pointed out an appreciable increase in CAT activity in Blue Mussel Mytilus spp. exposed to a mixture of microplastics, but a lack of changes in ROS production. They explained this result by the sensitivity of exploited technique (flow cytometery) and/or the sampling time chosen for its measurement. However, polystyrene microplastics in juvenile Eriocheir sinensis and larval zebrafish significantly alleviated the activity of CAT [51, 52]. In contrast, in a study involving Mytilus galloprovincialis, Sendra [53] reported a decreased percentage of ROS as a result of exposure to different sizes of PS-NPs.
SOD, the pioneering detoxifying enzyme, catalyzes the breakdown of ROS-generating superoxide anion (O2-) and is a key component within the primary defense system against oxidative stress-induced damage.
Lactate is cleared from the blood, primarily by the liver and, to a lesser extent, by the kidneys and skeletal muscles. Hyperlactatemia and lactic acidosis, a serious and sometimes life-threatening condition, is reflected by imbalance between the systemic generation of lactate and its clearance, or a combination of both. However, all of the available literature have found that the lipase or lactate levels in living organisms did not significantly change when exposed to micro/nano plastics [54, 55]. Although, ecotoxicological data on blood levels of lactate under nano(micro)plastics exposure are scarce, Karami et al. [54], have reported no negative impact of pristine or phenanthrene-loaded polyethylene fragments on African catfish.
The analysis of LDH provides a clearer picture whether - and to what extent - the energy metabolism was disrupted following exposure experiments. LDH activity has been widely accepted as a good biomarker of alterations in energy pathways induced by xenobiotics [56].
The observed positive associations between PS nanoplastics and a concurrent dose-dependent increase in creatinine and uric acid levels could be explained by dose-response kidney deterioration. Some studies in rats have reported increased creatinine concentrations as a consequence of kidney damage compared with control subjects [57]. It is somewhat surprising that translocation of fluorescent polystyrene nanoparticles from gastrointestinal tract to kidney has been previously reported in rats [58], and also possibly in our work. Increased levels of creatinine have also been reported in common carp (Cyprinus carpio) exposed to sub-lethal concentrations of microplastic and/or paraquat [34]. The increase in serum levels of urea provides further evidence for the toxicity of PS-NPs to the liver, as urea is the end-product of protein catabolism, which is in good agreement with proteins concentrations. Wright and Kelly [59] also revealed that MPs can accumulate and exert dose-dependent localized-particle toxicity by inducing inflammation and immune mechanisms in human.
As a cellular membrane enzyme, ALP catalyzes the dephosphorization of nucleotides, proteins, and alkaloids at an alkaline pH [54]. This enzyme may be liberated into the plasma because of the degeneration and necrosis of hepatocytes following exposure to NPs [60]. The increased activity of ALP in exposed animals may be speculated by damage to the membrane of liver cells, bile ducts, intestine mucous lining, and the renal tract [54].
The impact of microplastics at high dosages on common carp (Cyprinus carpio) (at 2 mg L− 1) [34] and amazonian cichlid (at 200 µg L− 1) [55], on ALP activity has been studied, demonstrating decreased ALP activity; but the impact of nano-scale plastic particulates exposure particularly on mammalians is still unknown. Karami et al., [54] also reported no magnification or diminution in plasma ALP activity of African catfish (Clarias gariepinus) because of exposure to LDPE MPS. By and large, these studies reflected some dearth in the digestive capabilities of exposed animals.
GGT is usually the first hepatic enzyme to rise in the blood as a consequence of bile ducts obstruction, which take bile from the liver to the intestines. Accordingly, it is the most sensitive bio indicator n the body for detecting bile duct troubles. Nevertheless, the GGT analysis is not specific candidate for differentiating between various causes of liver damage, as it may be increased by many types of liver diseases [54, 61], which is in line with visible accumulation of labeled PS NPs in the liver (Fig. 7). However, GGT coupled with other tests provides a lot of useful advice in determining the plausible cause of elevated ALP levels. To test whether the increment in ALP is corresponded to bone tissue damage, we assessed the serum levels of GGT. As both GGT and ALP levels shows increasing trends by exposure dosage, we speculate that the liver is damaged. If only ALP had increased, it would have indicated bone tissue damages. Therefore, GGT was used as a follow up to an elevated ALP to help in determining the damaged liver or tissue the bile ducts. This was in line with ROS (Fig. 2), where an increase in nanoplolystyrene doses is associated with oxidative stress that is a strong inducer of ALP in various tissues [62]. Perturbations in plasma GGT activity was also observed in common carp (Cyprinus carpio) where its activity decreased when exposed to micro-plastic particles [34].
Albumin and globulin decrement may also be assigned to the reduced protein synthesis in the liver of NPs-exposed animals. Decreased albumin levels may indeed result in osmoregulation defaults in blood [54]. The observed dose-dependent decreasing pattern in globulin levels could be attributed to the reduced immunity in exposed animals [63, 64]. The high ratio of A/G points towards the plausible liver and kidney tissues deterioration, which is in good agreement with the results of poor liver function. In line with our findings, exposure to MPs had induced alterations in the levels of total protein, globulin, and A/G ratio of African catfish (C. gariepinus), common carp (Cyprinus carpio), Nile Tilapia (Oreochromis niloticus) and gilthead seabream (Sparus aurata L.) [34, 54, 65].
The large effect size with meaningful changes put forward the generation of O2− may act a prime cause of oxidative impairment. Since µ0 is inside the interval, it was not possible to draw a meaningful conclusion relating to the increased activity of SOD in the present study and thus this result should be again repeated with higher sample size or meta-analysis should be done. However, the plausible inflammation and destabilization of lysosomal membranes could also be occurred [47]. To our knowledge, to date only one study has pointed out the toxic effects of nano(micro)plastics on SOD activity in rodent species [29]. Compared to the negative control and tris (2-chloroethy) phosphate (TCEP) alone, the binary mixture of PS-MPs and TCEP in that study induced the lowest activity in mice (Mus musculus). Rodent studies, however, have demonstrated alterations in SOD activity due to engineered nanoparticles exposure early in life or in adulthood [40, 66, 67]. Likewise, in the intestine of male marine medaka (Oryzias melastigma), chronic exposure to PS particles (10 µm) led to increased activity of SOD, while in females its activity was significantly decreased in ovaries [68]. Additionally, PS microbeads caused remarkable increase of SOD activity in zebrafish Danio rerio [5]. In this context, Ribeiro et al. [47] worked on the effects of clam Scrobicularia plana exposure to PS-MPs on oxidative stress biomarkers. From the battery of biomarkers (SOD, CAT, GST and GSH), only SOD was marked as the most prominent, other parameters did not show clear patterns of change. Given the conflicting data reported, clarity on the perturbations in SOD activity from exposure to nano(micro)plastics awaits outcome of further studies.
It has been proved that GPX detoxifies hydrogen peroxide or organic hydroxy peroxides through consumption of GSH. Meanwhile, GSH is used as a cofactor or substrate for GST (glutathione -S-transferase) [69]. Alternatively, associations might be explained by a combination of oxidative stress and enzymatic activities of antioxidant defense. The literature on associations between exposure to nano(micro)plastics and GSH content in aquatic organisms is not conclusive. According to Jeong et al., 2016 [70], for example, a toxicity study of fluorescently labeled polystyrene microbeads with different sizes (0.05, 0.5, and 6 µm) on Monogonont Rotifer (Brachionus koreanus) showed increased GSH content after 12 h in a size-dependent manner. Contrary to this study, however, declined GSH content has been reported in larval zebrafish [51, 71] and juvenile Eriocheir sinensis [52] exposed to MPs. Overall, there are diverging results concerning GSH content and evidence on the relationship between GSH content and oxidative stress are not sufficient.
Acetylcholinesterase, also known as acetylhydrolase, is the primary cholinesterase in the body, catalyzing the breakdown of acetylcholine and of some other choline esters that function as neurotransmitters. An analysis of the data available in the literature already confirms the inhibition of AChE as well as some other cellular antioxidant defense system by ROS [72]. Given the involvement of acetylcholinesterase in neurological functions including some physiological (e.g. growth, reproduction) and behavioural (e.g. memory) processes and irrespective of the mechanism leading to AChE inhibition, the anticholinesterase poisoning of nano-sized PS particulates in rodent species, commonly exploited for toxicity studies on which to base risk assessment for humans, provides an early warning sing of impacts at higher levels (e.g. organism, population). Nonetheless, the first evaluation of polystyrene NPs toxicity on the same subject demonstrated subtle and transient behavioral consequences in adult rats [26], denoting that neurotransmission might not be directly connected to the barely perceptible behavioral disorders. In support of our findings, Wei Lin et al. [73] observed declined AChE activity in Daphnia magna following exposure to various functionalized PS-NPs (PS-p-NH2, PS-n-NH2 and PS-COOH). They also reported a moderately positive but insignificant association between exposure to pristine plain PS-NPs and AChE activity.
A recent study by Ding et al., [74] tried to assess the AChE activity of red tilapia (Oreochromis niloticus) exposed to 0.1 µm PS-MPs. They investigated different concentrations of polystyrene nanoparticles (1, 10 and 100 µg L− 1) with adult fish for 14 consecutive days. There observed statistically significant reductions in acetylcholinesterase activity in fish brains exposed to PS particulates at all studied concentrations with maximum inhibition rate of 37.7%. Furthermore, although there was significant difference between the control and all exposed groups, no appreciable differences in AChE activity among samples treated with PS-NPs was perceived.
Likewise, in the study of Oliveira et al., [30] the mixture of Red polyethylene microspheres and pyrene induced the highest toxicity in juveniles of Pomatoschistus microps in terms of AChE activity inhibition, compared to MPs alone.
Cell membranes damage will indeed wield causal elevated levels of LDH in the blood [39]. In mice treated by polystyrene microplastics, Deng et al. [29] also observed an increasing trend in liver LDH activity with MPs exposure (p < 0.05). Likewise, in Amazonian cichlid, significantly higher LDH concentrations were observed after polystyrene MPs exposure [55]. In contrast, Karami et al. [54] found no association between activation of this enzyme and virgin microplastics exposure in African catfish. In a study on common carp (C. carpio) with and without exposure to microplastic particles, Haghi and Banaee [34] did not notice a meaningful LDH activity. Similar results have also been reported in literature, demonstrating depletion in energy production as a consequence of microplastics uptake both in toxicological and ecotoxicological studies [5, 34, 75, 76]. Nevertheless, evidence regarding the effects of plastic particulates exposure and particularly nanoplastics on energy metabolism is not conclusive.
Some studies have reported altered triglycerides concentration in the blood [34, 77], while, a lack of changes in serum levels of triglycerides has also been reported [78]. Triglycerides are needed to maintain the energy balance. In fact, Liver is the pioneering organ regulating the metabolism of triglycerides [79]. Accordingly, the increased circulatory triglyceride levels of exposed animals in our study could be assigned to liver lesions [54, 80] or plausible disturbance in its uptake in the intestinal tract [81].
In order to test whether the observed interruption in energy metabolism with PS-NPs exposure was correlated with oxidative stress, we conducted additional analyses where we noticed ROS content were positively correlated with glucose (r = 0.67, p = 0.002), triglycerides (r = 0.63, p = 0.004), lactate (r = 0.71, p = 0.001) as well as lactate dehydrogenase (r = 0.67, p = 0.002), demonstrating that elevated levels of these parameters did induce dysfunction and apoptosis in body, possibly through the formation of reactive oxygen species. However, studies underlying the relationship between energy metabolism and oxidative stress following exposure to nano(micro-)plastics is still extremely scarce.