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No-observed-adverse-effect level of hair pyrrole adducts in chronic n-hexane intoxication in rats
Neurotoxicology 78 (2020) 11–20
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Full Length Article
No-observed-adverse-effect level of hair pyrrole adducts in chronic n-hexane intoxication in rats
T
Xianjie Lia, Lulu Jianga,b, Ting Yua, Ming Lia, Qiong Wanga, Zhidan Liua, Keqin Xiea,* a b
Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, 02118, United States
ARTICLE INFO
ABSTRACT
Keywords: n-Hexane 2,5-Hexanedione Pyrrole adduct Intoxication Neuropathy No-observed-adverse-effect level
n-Hexane has been reported to induce serious peripheral neuropathy in workers. Pyrrole adducts are the unique reaction products of n-hexane in organisms and have been demonstrated to be critical to n-hexane neuropathy. Our previous studies have demonstrated that pyrrole adducts could accumulate in hair and showed high correlation with neuropathy at the end of experiments in rat models. In the present study, we examined the time course of hair pyrrole adducts and behavioral changes in rats exposed to different dosages of n-hexane in both treatment (24 weeks) and recovery phases. Our results showed: 1. After treatment, 1.0, 2.0, and 4.0 g/kg dosage groups all lost weight, but the 0.5 g/kg dosage group showed no impairment; after recovery, all impaired rats regained weight. 2. After treatment, 1.0, 2.0, and 4.0 g/kg dosage groups all showed a rise in gait scores, decreased rotarod latency, and decreased motor nerve conduction velocity, whereas the 0.5 g/kg dosage group showed no impairment; after recovery, all impaired rats were completely rehabilitated. 3. After treatment, levels of pyrrole adducts in serum, urine, and hair of experimental groups increased; after recovery, serum and urine pyrrole adducts showed no difference from the control (P > 0.05), whereas hair pyrrole adducts were significantly different from the control (P < 0.01). 4. The half-lives of serum and urine pyrrole adducts were 47.8–78.0 h and 42.7–52.9 h, while the half-life of hair pyrrole adducts was 14–24 weeks. 5. During treatment and recovery, levels of serum, urine, and hair pyrrole adducts showed high correlation with gait scores (P < 0.01), and hair pyrrole adducts had the largest partial correlation coefficient. In conclusion, hair pyrrole adducts could serve as a stable and reliable biomarker for the prevention of n-hexane intoxication. Furthermore, the no-observed-adverse-effect level of hair pyrrole adducts in rats is 275.2 ± 61.5 nmol/g protein. Further studies are required for the definition of the biological exposure limit in humans.
1. Introduction n-Hexane is a straight chain, saturated hydrocarbon obtained from certain petroleum fractions after various thermal or catalytic cracking steps (Ware, 1988). The main industrial applications of n-hexane include its use as a rubber and adhesive solvent in shoe factories and for the extraction of soybean oil, callous seed oil, and flaxseed oil (Chang et al., 1993). Although n-hexane itself has low toxicity, its metabolite, 2,5-hexanedione (2,5-HD), can induce several peripheral neuropathies. Clinical manifestations of 2,5-HD intoxication might begin with numbness and a tingling sensation in the toes and fingers, followed by progressive weakness and areflexia in the distal limbs, and sometimes even paralysis (Decaprio, 1987; Huang, 2008; Kutlu et al., 2009). Although it has been demonstrated for decades that n-hexane could induce peripheral neuropathy, n-hexane intoxication continues to occur
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occasionally, and efficient methods of prevention and clinical treatments are still lacking (Sendur et al., 2009). Common monitoring of nhexane exposure by gaseous sampling can only evaluate the concentration of n-hexane in the working environment, while not reflecting the accumulation of n-hexane in workers. Moreover, although the commonly used nerve injury biomarkers, including S100 calciumbinding protein B, neuron-specific enolase, nerve fibers, and myelin basic protein, could reflect the nerve injury, these biomarkers are not available in the early stages of pathogenesis. Although neuron-muscle electrophysiology tests can reflect the neuropathy of patients at a relatively early stage, the majority of patients do not go to the hospital until the symptoms are severe (Neghab et al., 2012; Wang et al., 2014), which makes the early detection of n-hexane intoxication infeasible. Therefore, it is of vital importance to develop an efficient surveillance method for n-hexane exposure and occupational intoxication.
Corresponding author. E-mail address: [email protected] (K. Xie).
https://doi.org/10.1016/j.neuro.2020.02.002 Received 26 June 2019; Received in revised form 18 January 2020; Accepted 3 February 2020 Available online 08 February 2020 0161-813X/ © 2020 Published by Elsevier B.V.
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In 2004, the American Conference of Governmental Industrial Hygienists set a biological exposure limit (BEL) for 2,5-HD (without hydrolysis) at no more than 0.4 mg/l in urine collected at the end of the workweek. However, Claudia’s study showed that exposure to n-hexane is different for various industries, and levels of 2,5-HD in urine are predominantly dependent on the type of the operation (Santos et al., 2002). Besides, 2,5-HD has a short half-life (20–30 h) and can react with the ε-amino group of lysine residues to form pyrrole adducts in vivo (Yin et al., 2013). Urinary 2,5-HD could also be converted to 2,5-dimethylpyrrole and interfere with the measurement of 2,5-HD (Ogata et al., 1991). Therefore, researchers began to study the relationship between pyrrole adducts and n-hexane intoxication (Graham et al., 1982; Decaprio et al., 1982; Jackowski, 1988). It has been reported that the level and rate of generation of pyrrole adducts in the nervous system are highly correlated to the pathogenesis induced by n-hexane or 2,5-HD (Genter et al., 1987; Sayre et al., 1986). Moreover, our previous studies that used diallyl trisulfide to block the bioactivation of n-hexane catalyzed by cytochrome P450 family 2 subfamily E member 1 (CYP2E1) successfully attenuated nerve impairment by reducing the formation of both 2,5-HD and the pyrrole adducts (Wang et al., 2017a). Hence, pyrrole adducts are thought to be promising biomarkers for the early detection and diagnosis of n-hexane-induced neuropathy. Many studies have shown that the concentrations of pyrrole adducts in serum and urine were significantly increased after n-hexane or 2,5HD exposure, and there is a high correlation between this increase and neuropathy (Torres et al., 2014a). Gaku’s study also demonstrated that blood protein-pyrrole adducts may serve as surrogates for pyrroles within the nervous system of workers and as biomarkers of effect as well as exposure (Ichihara et al., 2019). However, the half-life of pyrrole adducts in serum and urine was 60–80 h and dropped drastically a few days after exposure (Yin et al., 2014a). Therefore, this study aimed to find biomarkers with longer half-life that could reflect the cumulative exposure to n-hexane and exhibit the symptoms. Hair samples have long been used to detect the accumulation of illegal drugs and toxins in forensic tests as hair grows slowly (approximate rate of 0.25 cm/week) and possibly retains drugs and toxins permanently (Poon et al., 2014; Mupunga et al., 2017; Erickson et al., 2017). Johnson DJ and Lack L had reported the accumulation of pyrrole adducts in the hair of rats treated with 2,5-HD, although quantification analysis was not established at that time (Johnson et al., 1995; Lack et al., 2008). Our previous studies have demonstrated that pyrrole adducts could accumulate in hair in vivo and in vitro (Li et al., 2018). Pyrrole adducts in hair, serum, and urine at the end also showed a high correlation to neuropathy induced by n-hexane or 2,5-HD. In the present study, timedependent changes in the pyrrole adducts were detected, and behavioral performance was observed in a rat model, exposed to different doses of n-hexane, in both the treatment and recovery phases. Furthermore, the partial correlation coefficients and half-lives of pyrrole adducts in hair, serum, and urine were compared. The level of hair pyrrole adducts at which the maximum dose of n-hexane did not induce neuropathy was set as the no-observed-adverse-effect level (NOAEL) as the reference value for biological exposure limit in humans.
diluting the solution to 250 ml with distilled water. 2.2. Animal treatments All experiments were conducted in accordance with the National Institutes of Health NIH Guide for the Care and Use of Laboratory Animals and the principles in the “Use of Animals in Toxicology” and were approved by the Ethics Committee of Shandong University Institute of Preventive Medicine Permit Number: 20130801. Sixty-four male Wistar rats, 7 weeks old and weighing 210–220 g, were provided by the Experimental Animal Center of Shandong University. All rats were accommodated at 22 ± 2 °C and 50 ± 10 % relative humidity in an animal room with a 12 h light/dark cycle. Drinking water and commercial animal feed were available ad libitum. After acclimation, 40 rats were randomly divided into five groups (8 rats in each group): one control group and four experimental groups (0.5, 1.0, 2.0, and 4.0 g/(kg∙day), orally); four rats were housed per cage. A solution of n-hexane dissolved in corn oil was administered to rats in the experimental groups seven times per week, while control group rats received an equal volume of vehicle. Every rat in each group was continuously treated until the rat underwent paralysis, and all treatments were terminated by the end of the 24th week; the recovery state of the impaired rats was observed. After acclimation, 24 additional rats were divided evenly into four experimental groups as satellite groups for toxicokinetic studies; three of these rats were housed per cage. During the treatment process, the measurement of pyrrole adduct concentrations in the serum, urine, and hair was conducted every 2 weeks. Behavioral measurement was conducted once a week. The measurement of pyrrole adducts and neurobehavioral indices continued during the recovery phase. A dose of 1.0 g/kg of urethane was administered intraperitoneally (IP) to anesthetize the rats. Blood samples were collected 3–4 min after the injection after ensuring that the rats were properly anesthetized, as evidenced by the absence of the hind limb withdrawal reflex. By the end of the recovery phase, all rats were sacrificed after being anesthetized with urethane by hemospasia from the abdominal aorta. 2.3. Behavioral performance 2.3.1. Gait score evaluation Rats were placed in an open field and observed for 3 min. After observation, gait scores were evaluated, ranking from 1 to 4 (Song et al., 2012; Lopachin et al., 2005). Specifically, 1 = normal, unaffected gait; 2 = slightly abnormal gait (hind limbs showed uncoordinated placement, exaggerated or overcompensated movements, or were splayed slightly); 3 = moderately abnormal gait (obvious movement abnormalities characterized by markedly splayed hind limbs, ataxia, swaying, rocking, lurching, stumbling); 4 = severely abnormal gait (flat foot walk, hind limbs flat on the surface, crawling, or unable to support weight). The assessment of gait score was conducted in a blind manner.
2. Materials and methods
2.3.2. Rotarod latency test Rotarod latency was measured using rotarod equipment (ZS-ROM, Beijing Zhongshidichuang Technology and Development Co., Ltd, Beijing, China). As described previously, all rats received training before intoxication, which required the rats to stay on the equipment for at least 60 s at a velocity of 8 rpm (Monville et al., 2007). During the formal test, the original velocity was set at 0 rpm and accelerated smoothly to 40 rpm within 200 s. The length of time that each animal stayed on the rod was recorded as its latency to fall and was registered automatically by a trip switch.
2.1. Chemicals and reagents Sodium hydrate, ethanol, hydrochloric acid, guanidine chloride, and n-hexane (> 98 %) were purchased from Hongyan Reagent Factory (Tianjin, China). Trypsin, 4-dimethylaminobenzaldehyde (DMAB), and 2,5-dimethylpyrrole were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin and bicinchoninic acid (BCA) protein quantification kits were purchased from Thermos Fisher Scientific (Waltham, MA, USA). Detergents for washing rat hair were purchased from a nearby supermarket. Ehrlich’s reagent was prepared by dissolving 2.5 g of DMAB in 25 ml of concentrated hydrochloric acid and
2.3.3. Motor nerve conduction velocity measurement The motor nerve conduction velocity (MCV) of the rat tail was 12
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Fig. 1. Weight changes. A normal increase in body weight was notably retarded in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. *P < 0.05, **P < 0.01 vs control. By the end of recovery, all rat recovered to the level of control.
measured with an MD-3000 Biosystem, as described previously (Takeuchi and Sugiura, 1980). The electrodes were stainless steel needles 0.34 mm in diameter and approximately 15 mm long. The rat was fastened, and its tail was exposed to a pair of stimulating electrodes, which were connected to the two pairs of sense electrodes and an earth electrode. An electric stimulus of 5 V was applied by the stimulating electrodes, and two action potential oscillogram curves were obtained. The time between the two peak points and the distance between the two negative sense electrodes were recorded to calculate the MCV.
added to 6.6 ml of NaOH (1.2 mol/L) and heated in a 55 ℃ water bath for 1 h (Li et al., 2018). Supernatant (480 μL) was collected after centrifugation. Then, 20 μL of concentrated hydrochloric acid and 500 μL of 0.1 % trypsin were added and heating in the water bath continued for one more hour. The optical densities were determined spectrophotometrically at a wavelength of 526 nm, and pyrrole adduct concentrations were calculated based on the working curve generated with the co-digestion of hair and 2,5-DMP. Then, the BCA kit was used to determine the protein concentration in the solvent, and the final concentrations of the hair pyrrole adducts were presented as nmol/g protein.
2.4. Pyrrole adduct measurements
2.5. Toxicokinetic studies
According to Yin’s and Wang’s studies (Yin et al., 2014b; Wang et al., 2017b), blood samples were collected through the jugular vein 24 h after the last dose of n-hexane. Blood samples of 1 ml were collected with a 1 ml syringe. Serum samples were separated by centrifugation at 18,000 g for 10 min. A mixture containing 50 μL of serum and 50 μL of guanidine chloride (8 mol/L) was added to 96-well plates to measure the optical density (OD1) at 526 nm using an automatic microplate reader (Infinite 200Pro, Tecan Inc. Switzerland) (Yin et al., 2014b; Wang et al., 2017b). Then, 50 μL of Ehrlich’s reagent was added to obtain the OD2 value at 526 nm. The concentrations of the pyrrole adducts were calculated in terms of the changes in the OD values, referring to a standard working curve that was generated by using 2,5dimethylpyrrole (2,5-DMP) in the range of 0–60 nmol/mL. Urine samples were collected by tender stimulation at 24 h postdosing, and a minimum of 200 μL of urine was collected from each rat each time. After centrifugation, the supernatants were used to detect the concentrations of pyrrole adducts, as described above. The concentrations were calculated by the changes in the OD values, referring to a standard working curve that was generated with 2,5-DMP in the range of 0–360 nmol/mL. Hair samples (approximately 200 mg per sample from each rat) were collected with an electric razor, and the samples were completely washed and defatted. As described previously, 100 mg of hair was
The toxicokinetic studies of n-hexane consisted of two parts: the toxicokinetic change in pyrrole adducts at 5 days after a single dose of n-hexane and the clearance of the pyrrole adducts during the recovery phase after the end of the treatment. The satellite groups were treated in the same way as the experimental groups. In the 7th week, the rats in the satellite groups were fasted overnight with free access to water. A single administration of n-hexane was given to the rats at different doses. Samples of serum, urine, and hair were harvested from each rat at 1, 4, 8, 12, 24, 48, 72, 96, and 120 h after n-hexane administration. The pyrrole adduct concentrations were subsequently determined and analyzed by DASforeCDM 3.0 to calculate the area under the curves and the half-lives. Furthermore, the data from the serum, urine, and hair pyrrole adduct surveillance during the recovery phase were analyzed by DASforeCDM 3.0 to calculate the excretion half-lives. 2.6. Data analysis Data are presented as the mean ± standard deviation (SD) after analysis using SPSS 19.0 software. For all data, one-way analysis of variance (ANOVA) was performed, followed by Dennett’s post-hoc test. Multiple linear regression analyses were performed to determine the 13
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Fig. 2. Neuro-behavioral indices. Rising in gait score, decreasing of latency and decreased MCV were observed in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. By the end of recovery, all rat recovered.
gold standard behavioral index, and Pearson’s correlation coefficients and partial correlation coefficients were then calculated. The level of statistical significance was set at P < 0.05. The maximum concentration (Cmax) and the minimum concentration (Cmin) were obtained from the observed data. The area under the concentration-time curve (AUC(0-t)), the time to maximum concentration (Tmax), and half-life (t1/2) were calculated using DASforeCDM 3.0 software.
compared to the rats in the control group, suggesting a time-dependent gait disturbance in the 14th, 6th, and 3rd week of n-hexane intoxication, respectively (P < 0.01). At the end of the treatment, the rate of paralysis in the 1.0, 2.0, and 4.0 g/kg groups were 12.5 %, 100 %, and 100 %, respectively, whereas no paralysis was observed in the 0.5 g/kg group (Fig. 2A). The gait disturbance of rats in the 1.0, 2.0, and 4.0 g/ kg dose groups was gradually reversed by the 8th, 11th, and 12th week, respectively (Fig. 2B). All gait scores of the impaired rats returned to 1 point by the 32nd week (Fig. 2C).
3. Results 3.1. Changes in body weight
3.2.2. Rotarod latency test During the treatment process, rats treated with 1.0, 2.0, and 4.0 g/ kg n-hexane exhibited significantly decreased rotarod latencies starting at the 14th, 6th, and 3rd week, respectively (P < 0.05). At the end of the treatment, the rotarod latency of the rats in the 1.0, 2.0, and 4.0 g/kg groups were 49.4 ± 14.2 s, 23.4 ± 15.5 s, and 13.4 ± 10.4 s, respectively, which were significantly different from that of the control group at 116.0 ± 17.2 s (P < 0.01). However, the rats in the 0.5 g/kg group showed latencies of 112.4 ± 20.2 s, which were comparable to those seen for the control animals (P > 0.05) (Fig. 2D). Moreover, the recovery phase was able to rehabilitate the treated rats so that the rotarod latency of the rats treated with 1.0, 2.0, and 4.0 g/kg n-hexane was comparable to that of the control rats at the 5th, 9th, and 10th week after exposure, respectively (Fig. 2E). All latency of the impaired rats returned to normal levels after the 29th week (Fig. 2F).
As shown in Fig. 1, a normal increase in body weight was notably retarded in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. The body weight gain of the rats at exposure doses of 1.0, 2.0, and 4.0 g/kg showed significant differences compared to the control group in the 4th, 2nd, and 1st weeks, respectively (P < 0.05). As the treatment continued, the body weights of rats exposed to 1.0, 2.0, and 4.0 g/kg n-hexane began to fall in the 14th, 9th, and 4th week, respectively. At the end of the treatment, which was the 24th, 24th, 14th, and 10th week for the 0.5, 1.0, 2.0, and 4.0 g/kg groups, the mean body weights of these same treatment groups were 86.4 %, 81.4 %, 84.6 %, and 72.3 % of the control, respectively. Nevertheless, all impaired rats regained body weight and showed no difference from the control by the end of the 32nd week. 3.2. Behavioral performance
3.2.3. Motor nerve conductive velocity measurement During the treatment phase, rats in the 1.0, 2.0, and 4.0 g/kg groups showed decreased MCV starting by the 14th, 6th, and 4th week, respectively (P < 0.05). At the end of the treatment, the MCVs of rats in the 1.0, 2.0, and 4.0 g/kg groups were 24.0 ± 9.2 m/s, 21.0 ± 6.1 m/
3.2.1. Gait score evaluation During the treatment process, the gait scores of the rats in the 1.0, 2.0, and 4.0 g/kg n-hexane-exposed groups robustly increased 14
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Fig. 3. Pyrrole adducts changes. Notable increases in serum, urine and hair pyrrole adducts were observed in rats exposed to n-hexane (0.5, 1.0, 2.0, and 4.0 g/kg). By the end of recovery, serum and urine pyrrole adducts in rat returned to the level of control, while hair pyrrole adducts level was still detectable. Fig. 4. Toxicokinetics study. Serum pyrrole adducts in rats treated with n-hexane reached their peak values 12 h after treatment. The half-life of serum pyrrole adducts was 62.9 ± 15.1 h. In addition, the pyrrole adducts in the urine reached their peak values 8–9 h after treatment, with a half-life of 47.8 ± 5.1 h. While the concentrations of pyrrole adducts in hair mainly increased by the end of the experiment.
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Table 1a Toxicokinetics study during 120 h. AUC(0-t)
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
T1/2(h)
SPAs
UPAS
HPAs
SPAs
638.6 ± 41.7 927.6 ± 40.7 1432.8 ± 26.7 2271.9 ± 116.0
1854.1 ± 90.5 3662.2 ± 382.7 5265.4 ± 700.9 13043.1 ± 1169.7
26693.9 33713.9 39482.1 48203.3
± ± ± ±
2093.3 1761.6 2033.9 2885.6
38.2 49.5 51.4 62.5
UPAS ± ± ± ±
14.4 18.3 15.2 19.4
29.5 32.5 37.5 91.6
± ± ± ±
HPAs 5.3 1.5 2.6 13.8
/ / / /
Table 1b Toxicokinetics study during 120 h. Cmax (nmol/ml (g.pro))
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
Cmin (nmol/ml (g.pro.))
SPAs
UPAS
HPAs
8.6 ± 3.2 13.3 ± 1.2 22.3 ± 1.0 37.6 ± 3.9
51.6 ± 3.4 78.8 ± 1.5 135.2 ± 9.6 258.7 ± 3.0
312.8 378.0 412.2 469.9
SPAs ± ± ± ±
68.4 28.2 32.3 43.4
3.2 4.2 5.1 9.0
± ± ± ±
0.4 0.1 1.1 1.6
UPAS
HPAs
1.5 ± 0.2 7.0 ± 0.3 12.5 ± 3.2 21.7 ± 0.4
244.4 336.6 362.8 416.4
± ± ± ±
87.8 57.6 52.7 61.1
Table 2a Toxicokinetics during the recovery phase.
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
AUC(0-t)
T1/2(h)
SPAs
UPAS
HPAs
1008.0 ± 134.4 2755.2 ± 588.0 6434.4 ± 856.8 10550.4 ± 957.6
4720.8 ± 456.9 19370.4 ± 7728.0 38220.0 ± 3376.8 78842.4 ± 7543.2
201112.8 429391.2 720232.8 737940.0
SPAs ± ± ± ±
40185.6 48938.4 58531.2 95692.8
100.8 134.4 117.6 151.2
UPAS ± ± ± ±
16.8 15.7 14.9 15.9
151.3 117.9 218.4 453.6
HPAs ± ± ± ±
11.5 16.0 33.6 201.6
3662.4 3679.2 3561.6 5863.2
± ± ± ±
487.2 974.4 957.6 1008.0
Table 2b Toxicokinetics during the recovery phase.
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
Cmax (nmol/ml (g.pro.))
Cmin (nmol/ml (g.pro.))
SPAs
UPAS
HPAs
4.6 ± 1.2 15.2 ± 5.9 40.8 ± 9.0 48.9 ± 8.2
22.3 ± 2.3 61.6 ± 32.2 136.4 ± 11.6 285.1 ± 67.3
241.6 364.2 513.9 528.4
SPAs ± ± ± ±
28.6 42.1 81.9 96.3
0.7 0.8 0.7 0.9
± ± ± ±
UPAS 0.0 0.1 0.2 0.1
1.1 0.9 0.8 1.9
± ± ± ±
HPAs 0.2 0.2 0.2 0.5
158.4 253.8 276.2 254.5
± ± ± ±
30.6 43.4 22.6 18.4
s, and 17.6 ± 5.1 m/s, which were 53.7 %, 55.2 %, and 39.2 % that of the control animals (43.7 ± 12.0 m/s), respectively. However, rats in the 0.5 g/kg group had an MCV of 41.7 ± 9.8 m/s, showing no difference from the control group (P > 0.05) (Fig. 2G). During the recovery phase, the MCVs of the rats exposed to 1.0, 2.0, and 4.0 g/kg nhexane had increased to the levels of the control animals after 7, 11, and 12 weeks, respectively (Fig. 2H). All MCVs of the impaired rats returned to normal levels by the 31st week (Fig. 2I).
exposed to n-hexane showed a significant increase in accumulation in a dose-dependent manner compared to the control. In the recovery phase, the pyrrole adducts could still be detected in the hair, although their levels had decreased. That is, even after 14 weeks after exposure, the pyrrole adducts were significantly higher in the hair of rats treated with 0.5, 1.0, 2.0, and 4.0 g/kg n-hexane, as compared to that of the control group (P < 0.01) (Fig. 3H).
3.3. Pyrrole adduct measurements
3.4. Toxicokinetic studies
As shown in Fig. 3A, the pyrrole adducts in rat serum of all experimental groups showed significant increases in a dose-dependent manner compared to the control (P < 0.01). In the recovery phase, pyrrole adducts in the sera of rats treated with 0.5, 1.0, 2.0, and 4.0 g/ kg of n-hexane returned to the levels of the control after 1, 2, 4, and 5 weeks of exposure, respectively, which also showed a dose-dependent relationship (Fig. 3B). As shown in Fig. 3D, the pyrrole adducts in the urine of rats in all experimental groups increased in a dose-dependent manner (P < 0.01). Increased levels of pyrrole adducts in the urine of rats treated with 0.5, 1.0, 2.0, and 4.0 g/kg n-hexane were eliminated during the recovery phase after 4, 6, 9 and 12 weeks, respectively (Fig. 3E). As shown in Fig. 3G, pyrrole adducts in the hair of rats
As shown in Fig. 4, serum pyrrole adducts in rats treated with nhexane reached their peak values 12 h after treatment. The half-life of serum pyrrole adducts was 62.9 ± 15.1 h (Table 1a). In addition, the pyrrole adducts in the urine reached their peak values 8–9 h after treatment, with a half-life of 47.8 ± 5.1 h (Table 1a). Interestingly, the concentrations of pyrrole adducts in hair mainly increased by the end of the experiment (Table 1b). The half-lives were then calculated with data collected during the recovery phase, and the results showed that the half-lives of the pyrrole adducts in serum, urine, and hair were 0.8 ± 0.1, 1.4 ± 0.6, and 24.9 ± 7.5 weeks, respectively (Table 2a). The maximum and minimum concentrations of serum, urine and hair pyrrole adducts were shown in Table 2b. 16
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that the rats were exposed to. The rats in the 0.5 g/kg n-hexane dose group showed no behavioral impairments or increase in the pyrrole adduct concentrations in the serum or urine, whereas the pyrrole adducts in hair reached the level of 238.4 ± 24.4 nmol/g protein. (C1). The rats in the 4.0 g/kg dose group showed neuroimpairments starting from the 3rd week, with hair pyrrole adducts reaching 332.7 ± 76.4 nmol/g protein, whereas the decrease in weight gain began in the 2nd week when the hair pyrrole adduct levels were 312.8 ± 98.6 nmol/g protein (C2). Therefore, the NOAEL was set as 275.2 ± 61.5 nmol/g protein (1/2 of (C1+C2)) after two weeks of exposure. 4. Discussion It has been decades since researchers found that n-hexane overexposure could induce peripheral neuropathy in workers. However, the specific treatments for n-hexane intoxication are not yet available, which makes the elucidation of potential preventative strategies very important. In the present study, hair pyrrole adducts were identified as an effective long-term biomarker of n-hexane exposure, and an NOAEL value of n-hexane intoxication was defined based on data from the rat model. These findings show great potential to help prevent occupational intoxication of n-hexane in the future. First, the dose-dependence of the change in weight gain in the rats treated with n-hexane was compared to that of the control animals. The results showed that weight gain of rats treated with n-hexane doses of 1.0, 2.0, and 4.0 g/kg had decreased by the end of the intoxication, whereas the comparison between the 0.5 g/kg dose group and the control showed no significant difference, which was consistent with our previous studies (Yin et al., 2014b). The weights of the affected rats all returned to levels comparable to those of the control animals at the end of the recovery phase. These results revealed that the doses of n-hexane that were used to establish the n-hexane intoxication model in rats were appropriate. To detect the phenotype of peripheral nerve impairment in the nhexane-treated rats, three standard parameters were measured throughout the treatment and recovery phases. The results showed that the onset of gait score changes and decrease in rotarod latency were observed first, followed by decrease in MCV. Moreover, during the recovery phase, it took a longer time for the MCV to be restored in the rats than for the muscles to recover, which was approximately 4 weeks. This time lag might suggest the relatively weak recoverability of the nervous system. The present study is the first to capture the spontaneous rehabilitation of peripheral nerve impairment induced by n-hexane in rats, which is consistent with case reports in humans showing that patients completely recovered after 1–2 years after exposure (Huang, 2008). Thus, these findings recapitulate and verify the phenomenon that n-hexane-induced peripheral neuropathy is reversible. After the establishment of the n-hexane intoxication model, we tried to identify the proper biomarkers for the assessment of the exposure level. 2,5-HD has a relatively short half-life (Yin et al., 2013), is not stable, and easily reacts with other substances to form pyrrole adducts (Torres et al., 2014b). Our previous study had demonstrated that concentrations of pyrrole adducts in the serum and urine of n-hexanetreated rats gradually increased in a dose-dependent manner during the treatment phase (Yin et al., 2014b). Therefore, in the current study, the levels of pyrrole adducts in rat serum, urine, and hair were determined in both the treatment and recovery stages. During the treatment phase, consistent with our previous report, our current results showed that the concentrations of pyrrole adducts in the serum, urine, and hair increased in a dose-dependent manner with n-hexane administration. However, during the recovery phase, the concentrations of pyrrole adducts in the serum and urine drastically dropped and returned to the levels of the control animals at the end of the recovery phase. These results suggest that pyrrole adducts in serum and urine had short half-lives and might not be able to serve as effective biomarkers.
Fig. 5. Correlation analysis. A strong correlation between the concentrations of the pyrrole adducts in serum, urine, and hair and the gait scores of both the treatment and recovery phases.
3.5. Correlation analysis and calculation of NOAEL As shown in Fig. 5, correlation analyses showed that there was a strong correlation between the concentrations of the pyrrole adducts in serum, urine, and hair and the gait scores of both the treatment and recovery phases. In the treatment phase (T), the concentrations of the pyrrole adducts in the serum, urine, and hair all showed strong correlation to the gait scores, with correlation coefficients of 0.701, 0.670, and 0.683, respectively (P < 0.01). A similar correlation was seen between the pyrrole adducts in the serum, urine, and hair of the rats exposed to n-hexane and their gait scores during the recovery phase (R), with correlation coefficients of 0.456, 0.570, and 0.702, respectively (P < 0.01). Moreover, there was strong correlation between the rotarod latency and MCV results and the gait scores in both phases (P < 0.01). In addition, multiple regression analysis was performed in which the behavioral indices were defined as Y, and the concentrations of serum, urine, and hair pyrrole adducts were defined as X (Fig. 6). The results showed that the multiple regression coefficients (multiple R) of the gait scores were the largest among the three indices in both the intoxication and recovery phases (Table 3a). Therefore, the gait score was set as the gold standard for the calculation of the correlation coefficients of each pyrrole adduct. he partial correlation coefficients were also calculated (Table 3b). In the treatment phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.193, 0.120, and 0.324, respectively, and in the recovery phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.050, 0.115, and 0.322, respectively. The accumulation of pyrrole adducts in hair continued with the growth of hair. Thus, we hypothesized that the pyrrole adducts in hair might be able to provide a real-time status of the total level of n-hexane 17
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Fig. 6. Mutiple regression analysis. In the treatment phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.193, 0.120, and 0.324, respectively, and in the recovery phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.050, 0.115, and 0.322, respectively.
Interestingly, in contrast to the serum and urine data, levels of pyrrole adducts in the hair of rats with n-hexane intoxication showed a slower and steady increase during the treatment phase as well as a long-term and sustained presence during the recovery phase. These results suggest that the levels of pyrrole adducts in hair could be a promising biomarker to monitor and diagnose n-hexane intoxication. Occupational monitoring of 2,5-HD set a BEL value at no more than 0.4 mg/l in urine
collected at the end of the workweek. However, as mentioned earlier, the short half-life and instability of 2,5-HD interfere with its monitoring. In contrast, our results showed that hair pyrrole adducts seemed to have longer half-lives and were much more stable. A quick digestion of hair by a combination of NaOH and trypsin, followed by spectrophotometric measurement could determine the pyrrole adduct concentrations. Although spectrophotometry is not as advanced as gas 18
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recovery were within the NOAEL value. Further studies are needed for the definition of the BEL value in humans.
Table 3a Multiple regression analysis results. Variables
PAs T PAs R
Multiple R
P value
Gait score
Rota-rod
MCV
Gait score
Rota-rod
MCV
0.746** 0.587**
−0.499** −0.450*
−0.443** −0.321#
0.000 0.000
0.000 0.003
0.000 0.086
5. Conclusions First, there was a strong positive correlation between the hair pyrrole adduct concentration and peripheral neuropathy, and the hair pyrrole adducts had higher partial correlation coefficients than serum and urine pyrrole adducts in both the treatment and recovery phases. Second, the pyrrole adducts in hair showed remarkable potential to be efficient biomarkers for the prevention and diagnosis of occupational nhexane intoxication because they had much longer excretion half-lives (21–34 weeks) than those of serum and urine. Third, based on our rat model, we suggest that hair pyrrole adduct levels of less than 275.2 ± 61.5 nmol/g protein be referred to as the NOAEL for n-hexane exposure.
*P < 0.05, **P < 0.01, #P > 0.05. Table 3b Correlation analysis ad partial correlation analysis. Variables
SPAs T UPAs T HPAs T SPAs R UPAs R HPAs R
Correlation analysis
Partial correlation analysis
Gait score
P value
Gait score
P value
0.701** 0.669** 0.693** 0.535** 0.601** 0.702**
0.000 0.000 0.000 0.000 0.000 0.000
0.193** 0.120* 0.324** −0.005# 0.241** 0.488**
0.000 0.019 0.000 0.700 0.001 0.000
CRediT authorship contribution statement Xianjie Li: Project administration, Methodology, Formal analysis, Software, Visualization, Writing - original draft. Lulu Jiang: Writing review & editing. Ting Yu: Project administration. Ming Li: Project administration. Qiong Wang: Project administration. Zhidan Liu: Project administration. Keqin Xie: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.
*P < 0.05, **P < 0.01, #P > 0.05.
chromatography, it enables a rapid screening measurement and a longterm surveillance every single week. The toxicokinetic results showed that pyrrole adduct concentrations in serum and urine reached their peak values at 12 h and 8 h, respectively, and then began to fall, whereas the concentrations of the hair pyrrole adducts increased slightly during the five-day test. The halflives of the hair pyrrole adducts could not be calculated during the fiveday test because the hair pyrrole adducts were detectable for a much longer time. Thus, we used the data from the recovery phase to calculate the excretion half-lives of the hair pyrrole adducts when the chronic exposure was discontinued. The excretion half-lives of the pyrrole adducts in the serum and urine were approximately 100–151.2 h (0.7–0.9 week) and 117.6–453.6 h (0.7–2.7 weeks). In contrast, the half-life of the hair pyrrole adducts was almost 3561.6–5947.2 h (21.2–35.4 weeks). Thus, there was a strong positive correlation between the concentrations of the hair pyrrole adducts at the end and n-hexane induced peripheral neuropathy. These results further demonstrate that hair pyrrole adducts have the potential to evaluate the peripheral nerve system status of organisms, regardless of the duration of exposure to n-hexane. In the present study, we analyzed the correlation between the levels of pyrrole adducts in the hair and peripheral neuropathy under different doses of n-hexane intoxication over a long time course. Results of the correlation analysis showed that pyrrole adducts in serum, urine, and hair all showed a strong, positive correlation to the gait scores, which was also suggested by previous studies (Yin et al., 2013; Wang et al., 2017a; Li et al., 2018). Notably, the pyrrole adducts in hair had higher partial correlation coefficients than urine and serum pyrrole adducts. Moreover, hair pyrrole adducts showed the strongest relationship with the onset and disappearance of peripheral nervous system impairments among the three biosamples. Our previous in vitro study also found that there was no difference between the rates of formation of pyrrole adducts in human and rat hair samples after treatment with 2.5-HD; hence the hair pyrrole adducts in workers might also accumulate in a similar manner and indicate the effects of exposure to n-hexane. Therefore, we chose the pyrrole adducts in the hair to establish the NOAEL value. Taking the final concentration of hair pyrrole adducts in rats of the 0.5 g/kg dosage group (238.4 ± 24.4 nmol/g protein) and the minimum concentration influencing the weight gain in the 4.0 g/kg dose group (312.8 ± 98.6 nmol/g.protein), the NOAEL was set as 275.2 ± 61.5 nmol/g protein after two weeks of exposure. Moreover, the levels of hair pyrrole adducts in impaired rats after
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Full Length Article
No-observed-adverse-effect level of hair pyrrole adducts in chronic n-hexane intoxication in rats
T
Xianjie Lia, Lulu Jianga,b, Ting Yua, Ming Lia, Qiong Wanga, Zhidan Liua, Keqin Xiea,* a b
Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, 02118, United States
ARTICLE INFO
ABSTRACT
Keywords: n-Hexane 2,5-Hexanedione Pyrrole adduct Intoxication Neuropathy No-observed-adverse-effect level
n-Hexane has been reported to induce serious peripheral neuropathy in workers. Pyrrole adducts are the unique reaction products of n-hexane in organisms and have been demonstrated to be critical to n-hexane neuropathy. Our previous studies have demonstrated that pyrrole adducts could accumulate in hair and showed high correlation with neuropathy at the end of experiments in rat models. In the present study, we examined the time course of hair pyrrole adducts and behavioral changes in rats exposed to different dosages of n-hexane in both treatment (24 weeks) and recovery phases. Our results showed: 1. After treatment, 1.0, 2.0, and 4.0 g/kg dosage groups all lost weight, but the 0.5 g/kg dosage group showed no impairment; after recovery, all impaired rats regained weight. 2. After treatment, 1.0, 2.0, and 4.0 g/kg dosage groups all showed a rise in gait scores, decreased rotarod latency, and decreased motor nerve conduction velocity, whereas the 0.5 g/kg dosage group showed no impairment; after recovery, all impaired rats were completely rehabilitated. 3. After treatment, levels of pyrrole adducts in serum, urine, and hair of experimental groups increased; after recovery, serum and urine pyrrole adducts showed no difference from the control (P > 0.05), whereas hair pyrrole adducts were significantly different from the control (P < 0.01). 4. The half-lives of serum and urine pyrrole adducts were 47.8–78.0 h and 42.7–52.9 h, while the half-life of hair pyrrole adducts was 14–24 weeks. 5. During treatment and recovery, levels of serum, urine, and hair pyrrole adducts showed high correlation with gait scores (P < 0.01), and hair pyrrole adducts had the largest partial correlation coefficient. In conclusion, hair pyrrole adducts could serve as a stable and reliable biomarker for the prevention of n-hexane intoxication. Furthermore, the no-observed-adverse-effect level of hair pyrrole adducts in rats is 275.2 ± 61.5 nmol/g protein. Further studies are required for the definition of the biological exposure limit in humans.
1. Introduction n-Hexane is a straight chain, saturated hydrocarbon obtained from certain petroleum fractions after various thermal or catalytic cracking steps (Ware, 1988). The main industrial applications of n-hexane include its use as a rubber and adhesive solvent in shoe factories and for the extraction of soybean oil, callous seed oil, and flaxseed oil (Chang et al., 1993). Although n-hexane itself has low toxicity, its metabolite, 2,5-hexanedione (2,5-HD), can induce several peripheral neuropathies. Clinical manifestations of 2,5-HD intoxication might begin with numbness and a tingling sensation in the toes and fingers, followed by progressive weakness and areflexia in the distal limbs, and sometimes even paralysis (Decaprio, 1987; Huang, 2008; Kutlu et al., 2009). Although it has been demonstrated for decades that n-hexane could induce peripheral neuropathy, n-hexane intoxication continues to occur
⁎
occasionally, and efficient methods of prevention and clinical treatments are still lacking (Sendur et al., 2009). Common monitoring of nhexane exposure by gaseous sampling can only evaluate the concentration of n-hexane in the working environment, while not reflecting the accumulation of n-hexane in workers. Moreover, although the commonly used nerve injury biomarkers, including S100 calciumbinding protein B, neuron-specific enolase, nerve fibers, and myelin basic protein, could reflect the nerve injury, these biomarkers are not available in the early stages of pathogenesis. Although neuron-muscle electrophysiology tests can reflect the neuropathy of patients at a relatively early stage, the majority of patients do not go to the hospital until the symptoms are severe (Neghab et al., 2012; Wang et al., 2014), which makes the early detection of n-hexane intoxication infeasible. Therefore, it is of vital importance to develop an efficient surveillance method for n-hexane exposure and occupational intoxication.
Corresponding author. E-mail address: [email protected] (K. Xie).
https://doi.org/10.1016/j.neuro.2020.02.002 Received 26 June 2019; Received in revised form 18 January 2020; Accepted 3 February 2020 Available online 08 February 2020 0161-813X/ © 2020 Published by Elsevier B.V.
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In 2004, the American Conference of Governmental Industrial Hygienists set a biological exposure limit (BEL) for 2,5-HD (without hydrolysis) at no more than 0.4 mg/l in urine collected at the end of the workweek. However, Claudia’s study showed that exposure to n-hexane is different for various industries, and levels of 2,5-HD in urine are predominantly dependent on the type of the operation (Santos et al., 2002). Besides, 2,5-HD has a short half-life (20–30 h) and can react with the ε-amino group of lysine residues to form pyrrole adducts in vivo (Yin et al., 2013). Urinary 2,5-HD could also be converted to 2,5-dimethylpyrrole and interfere with the measurement of 2,5-HD (Ogata et al., 1991). Therefore, researchers began to study the relationship between pyrrole adducts and n-hexane intoxication (Graham et al., 1982; Decaprio et al., 1982; Jackowski, 1988). It has been reported that the level and rate of generation of pyrrole adducts in the nervous system are highly correlated to the pathogenesis induced by n-hexane or 2,5-HD (Genter et al., 1987; Sayre et al., 1986). Moreover, our previous studies that used diallyl trisulfide to block the bioactivation of n-hexane catalyzed by cytochrome P450 family 2 subfamily E member 1 (CYP2E1) successfully attenuated nerve impairment by reducing the formation of both 2,5-HD and the pyrrole adducts (Wang et al., 2017a). Hence, pyrrole adducts are thought to be promising biomarkers for the early detection and diagnosis of n-hexane-induced neuropathy. Many studies have shown that the concentrations of pyrrole adducts in serum and urine were significantly increased after n-hexane or 2,5HD exposure, and there is a high correlation between this increase and neuropathy (Torres et al., 2014a). Gaku’s study also demonstrated that blood protein-pyrrole adducts may serve as surrogates for pyrroles within the nervous system of workers and as biomarkers of effect as well as exposure (Ichihara et al., 2019). However, the half-life of pyrrole adducts in serum and urine was 60–80 h and dropped drastically a few days after exposure (Yin et al., 2014a). Therefore, this study aimed to find biomarkers with longer half-life that could reflect the cumulative exposure to n-hexane and exhibit the symptoms. Hair samples have long been used to detect the accumulation of illegal drugs and toxins in forensic tests as hair grows slowly (approximate rate of 0.25 cm/week) and possibly retains drugs and toxins permanently (Poon et al., 2014; Mupunga et al., 2017; Erickson et al., 2017). Johnson DJ and Lack L had reported the accumulation of pyrrole adducts in the hair of rats treated with 2,5-HD, although quantification analysis was not established at that time (Johnson et al., 1995; Lack et al., 2008). Our previous studies have demonstrated that pyrrole adducts could accumulate in hair in vivo and in vitro (Li et al., 2018). Pyrrole adducts in hair, serum, and urine at the end also showed a high correlation to neuropathy induced by n-hexane or 2,5-HD. In the present study, timedependent changes in the pyrrole adducts were detected, and behavioral performance was observed in a rat model, exposed to different doses of n-hexane, in both the treatment and recovery phases. Furthermore, the partial correlation coefficients and half-lives of pyrrole adducts in hair, serum, and urine were compared. The level of hair pyrrole adducts at which the maximum dose of n-hexane did not induce neuropathy was set as the no-observed-adverse-effect level (NOAEL) as the reference value for biological exposure limit in humans.
diluting the solution to 250 ml with distilled water. 2.2. Animal treatments All experiments were conducted in accordance with the National Institutes of Health NIH Guide for the Care and Use of Laboratory Animals and the principles in the “Use of Animals in Toxicology” and were approved by the Ethics Committee of Shandong University Institute of Preventive Medicine Permit Number: 20130801. Sixty-four male Wistar rats, 7 weeks old and weighing 210–220 g, were provided by the Experimental Animal Center of Shandong University. All rats were accommodated at 22 ± 2 °C and 50 ± 10 % relative humidity in an animal room with a 12 h light/dark cycle. Drinking water and commercial animal feed were available ad libitum. After acclimation, 40 rats were randomly divided into five groups (8 rats in each group): one control group and four experimental groups (0.5, 1.0, 2.0, and 4.0 g/(kg∙day), orally); four rats were housed per cage. A solution of n-hexane dissolved in corn oil was administered to rats in the experimental groups seven times per week, while control group rats received an equal volume of vehicle. Every rat in each group was continuously treated until the rat underwent paralysis, and all treatments were terminated by the end of the 24th week; the recovery state of the impaired rats was observed. After acclimation, 24 additional rats were divided evenly into four experimental groups as satellite groups for toxicokinetic studies; three of these rats were housed per cage. During the treatment process, the measurement of pyrrole adduct concentrations in the serum, urine, and hair was conducted every 2 weeks. Behavioral measurement was conducted once a week. The measurement of pyrrole adducts and neurobehavioral indices continued during the recovery phase. A dose of 1.0 g/kg of urethane was administered intraperitoneally (IP) to anesthetize the rats. Blood samples were collected 3–4 min after the injection after ensuring that the rats were properly anesthetized, as evidenced by the absence of the hind limb withdrawal reflex. By the end of the recovery phase, all rats were sacrificed after being anesthetized with urethane by hemospasia from the abdominal aorta. 2.3. Behavioral performance 2.3.1. Gait score evaluation Rats were placed in an open field and observed for 3 min. After observation, gait scores were evaluated, ranking from 1 to 4 (Song et al., 2012; Lopachin et al., 2005). Specifically, 1 = normal, unaffected gait; 2 = slightly abnormal gait (hind limbs showed uncoordinated placement, exaggerated or overcompensated movements, or were splayed slightly); 3 = moderately abnormal gait (obvious movement abnormalities characterized by markedly splayed hind limbs, ataxia, swaying, rocking, lurching, stumbling); 4 = severely abnormal gait (flat foot walk, hind limbs flat on the surface, crawling, or unable to support weight). The assessment of gait score was conducted in a blind manner.
2. Materials and methods
2.3.2. Rotarod latency test Rotarod latency was measured using rotarod equipment (ZS-ROM, Beijing Zhongshidichuang Technology and Development Co., Ltd, Beijing, China). As described previously, all rats received training before intoxication, which required the rats to stay on the equipment for at least 60 s at a velocity of 8 rpm (Monville et al., 2007). During the formal test, the original velocity was set at 0 rpm and accelerated smoothly to 40 rpm within 200 s. The length of time that each animal stayed on the rod was recorded as its latency to fall and was registered automatically by a trip switch.
2.1. Chemicals and reagents Sodium hydrate, ethanol, hydrochloric acid, guanidine chloride, and n-hexane (> 98 %) were purchased from Hongyan Reagent Factory (Tianjin, China). Trypsin, 4-dimethylaminobenzaldehyde (DMAB), and 2,5-dimethylpyrrole were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin and bicinchoninic acid (BCA) protein quantification kits were purchased from Thermos Fisher Scientific (Waltham, MA, USA). Detergents for washing rat hair were purchased from a nearby supermarket. Ehrlich’s reagent was prepared by dissolving 2.5 g of DMAB in 25 ml of concentrated hydrochloric acid and
2.3.3. Motor nerve conduction velocity measurement The motor nerve conduction velocity (MCV) of the rat tail was 12
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Fig. 1. Weight changes. A normal increase in body weight was notably retarded in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. *P < 0.05, **P < 0.01 vs control. By the end of recovery, all rat recovered to the level of control.
measured with an MD-3000 Biosystem, as described previously (Takeuchi and Sugiura, 1980). The electrodes were stainless steel needles 0.34 mm in diameter and approximately 15 mm long. The rat was fastened, and its tail was exposed to a pair of stimulating electrodes, which were connected to the two pairs of sense electrodes and an earth electrode. An electric stimulus of 5 V was applied by the stimulating electrodes, and two action potential oscillogram curves were obtained. The time between the two peak points and the distance between the two negative sense electrodes were recorded to calculate the MCV.
added to 6.6 ml of NaOH (1.2 mol/L) and heated in a 55 ℃ water bath for 1 h (Li et al., 2018). Supernatant (480 μL) was collected after centrifugation. Then, 20 μL of concentrated hydrochloric acid and 500 μL of 0.1 % trypsin were added and heating in the water bath continued for one more hour. The optical densities were determined spectrophotometrically at a wavelength of 526 nm, and pyrrole adduct concentrations were calculated based on the working curve generated with the co-digestion of hair and 2,5-DMP. Then, the BCA kit was used to determine the protein concentration in the solvent, and the final concentrations of the hair pyrrole adducts were presented as nmol/g protein.
2.4. Pyrrole adduct measurements
2.5. Toxicokinetic studies
According to Yin’s and Wang’s studies (Yin et al., 2014b; Wang et al., 2017b), blood samples were collected through the jugular vein 24 h after the last dose of n-hexane. Blood samples of 1 ml were collected with a 1 ml syringe. Serum samples were separated by centrifugation at 18,000 g for 10 min. A mixture containing 50 μL of serum and 50 μL of guanidine chloride (8 mol/L) was added to 96-well plates to measure the optical density (OD1) at 526 nm using an automatic microplate reader (Infinite 200Pro, Tecan Inc. Switzerland) (Yin et al., 2014b; Wang et al., 2017b). Then, 50 μL of Ehrlich’s reagent was added to obtain the OD2 value at 526 nm. The concentrations of the pyrrole adducts were calculated in terms of the changes in the OD values, referring to a standard working curve that was generated by using 2,5dimethylpyrrole (2,5-DMP) in the range of 0–60 nmol/mL. Urine samples were collected by tender stimulation at 24 h postdosing, and a minimum of 200 μL of urine was collected from each rat each time. After centrifugation, the supernatants were used to detect the concentrations of pyrrole adducts, as described above. The concentrations were calculated by the changes in the OD values, referring to a standard working curve that was generated with 2,5-DMP in the range of 0–360 nmol/mL. Hair samples (approximately 200 mg per sample from each rat) were collected with an electric razor, and the samples were completely washed and defatted. As described previously, 100 mg of hair was
The toxicokinetic studies of n-hexane consisted of two parts: the toxicokinetic change in pyrrole adducts at 5 days after a single dose of n-hexane and the clearance of the pyrrole adducts during the recovery phase after the end of the treatment. The satellite groups were treated in the same way as the experimental groups. In the 7th week, the rats in the satellite groups were fasted overnight with free access to water. A single administration of n-hexane was given to the rats at different doses. Samples of serum, urine, and hair were harvested from each rat at 1, 4, 8, 12, 24, 48, 72, 96, and 120 h after n-hexane administration. The pyrrole adduct concentrations were subsequently determined and analyzed by DASforeCDM 3.0 to calculate the area under the curves and the half-lives. Furthermore, the data from the serum, urine, and hair pyrrole adduct surveillance during the recovery phase were analyzed by DASforeCDM 3.0 to calculate the excretion half-lives. 2.6. Data analysis Data are presented as the mean ± standard deviation (SD) after analysis using SPSS 19.0 software. For all data, one-way analysis of variance (ANOVA) was performed, followed by Dennett’s post-hoc test. Multiple linear regression analyses were performed to determine the 13
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Fig. 2. Neuro-behavioral indices. Rising in gait score, decreasing of latency and decreased MCV were observed in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. By the end of recovery, all rat recovered.
gold standard behavioral index, and Pearson’s correlation coefficients and partial correlation coefficients were then calculated. The level of statistical significance was set at P < 0.05. The maximum concentration (Cmax) and the minimum concentration (Cmin) were obtained from the observed data. The area under the concentration-time curve (AUC(0-t)), the time to maximum concentration (Tmax), and half-life (t1/2) were calculated using DASforeCDM 3.0 software.
compared to the rats in the control group, suggesting a time-dependent gait disturbance in the 14th, 6th, and 3rd week of n-hexane intoxication, respectively (P < 0.01). At the end of the treatment, the rate of paralysis in the 1.0, 2.0, and 4.0 g/kg groups were 12.5 %, 100 %, and 100 %, respectively, whereas no paralysis was observed in the 0.5 g/kg group (Fig. 2A). The gait disturbance of rats in the 1.0, 2.0, and 4.0 g/ kg dose groups was gradually reversed by the 8th, 11th, and 12th week, respectively (Fig. 2B). All gait scores of the impaired rats returned to 1 point by the 32nd week (Fig. 2C).
3. Results 3.1. Changes in body weight
3.2.2. Rotarod latency test During the treatment process, rats treated with 1.0, 2.0, and 4.0 g/ kg n-hexane exhibited significantly decreased rotarod latencies starting at the 14th, 6th, and 3rd week, respectively (P < 0.05). At the end of the treatment, the rotarod latency of the rats in the 1.0, 2.0, and 4.0 g/kg groups were 49.4 ± 14.2 s, 23.4 ± 15.5 s, and 13.4 ± 10.4 s, respectively, which were significantly different from that of the control group at 116.0 ± 17.2 s (P < 0.01). However, the rats in the 0.5 g/kg group showed latencies of 112.4 ± 20.2 s, which were comparable to those seen for the control animals (P > 0.05) (Fig. 2D). Moreover, the recovery phase was able to rehabilitate the treated rats so that the rotarod latency of the rats treated with 1.0, 2.0, and 4.0 g/kg n-hexane was comparable to that of the control rats at the 5th, 9th, and 10th week after exposure, respectively (Fig. 2E). All latency of the impaired rats returned to normal levels after the 29th week (Fig. 2F).
As shown in Fig. 1, a normal increase in body weight was notably retarded in rats exposed to n-hexane (1.0, 2.0, and 4.0 g/kg), but not in the 0.5 g/kg group or the control group. The body weight gain of the rats at exposure doses of 1.0, 2.0, and 4.0 g/kg showed significant differences compared to the control group in the 4th, 2nd, and 1st weeks, respectively (P < 0.05). As the treatment continued, the body weights of rats exposed to 1.0, 2.0, and 4.0 g/kg n-hexane began to fall in the 14th, 9th, and 4th week, respectively. At the end of the treatment, which was the 24th, 24th, 14th, and 10th week for the 0.5, 1.0, 2.0, and 4.0 g/kg groups, the mean body weights of these same treatment groups were 86.4 %, 81.4 %, 84.6 %, and 72.3 % of the control, respectively. Nevertheless, all impaired rats regained body weight and showed no difference from the control by the end of the 32nd week. 3.2. Behavioral performance
3.2.3. Motor nerve conductive velocity measurement During the treatment phase, rats in the 1.0, 2.0, and 4.0 g/kg groups showed decreased MCV starting by the 14th, 6th, and 4th week, respectively (P < 0.05). At the end of the treatment, the MCVs of rats in the 1.0, 2.0, and 4.0 g/kg groups were 24.0 ± 9.2 m/s, 21.0 ± 6.1 m/
3.2.1. Gait score evaluation During the treatment process, the gait scores of the rats in the 1.0, 2.0, and 4.0 g/kg n-hexane-exposed groups robustly increased 14
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Fig. 3. Pyrrole adducts changes. Notable increases in serum, urine and hair pyrrole adducts were observed in rats exposed to n-hexane (0.5, 1.0, 2.0, and 4.0 g/kg). By the end of recovery, serum and urine pyrrole adducts in rat returned to the level of control, while hair pyrrole adducts level was still detectable. Fig. 4. Toxicokinetics study. Serum pyrrole adducts in rats treated with n-hexane reached their peak values 12 h after treatment. The half-life of serum pyrrole adducts was 62.9 ± 15.1 h. In addition, the pyrrole adducts in the urine reached their peak values 8–9 h after treatment, with a half-life of 47.8 ± 5.1 h. While the concentrations of pyrrole adducts in hair mainly increased by the end of the experiment.
15
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Table 1a Toxicokinetics study during 120 h. AUC(0-t)
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
T1/2(h)
SPAs
UPAS
HPAs
SPAs
638.6 ± 41.7 927.6 ± 40.7 1432.8 ± 26.7 2271.9 ± 116.0
1854.1 ± 90.5 3662.2 ± 382.7 5265.4 ± 700.9 13043.1 ± 1169.7
26693.9 33713.9 39482.1 48203.3
± ± ± ±
2093.3 1761.6 2033.9 2885.6
38.2 49.5 51.4 62.5
UPAS ± ± ± ±
14.4 18.3 15.2 19.4
29.5 32.5 37.5 91.6
± ± ± ±
HPAs 5.3 1.5 2.6 13.8
/ / / /
Table 1b Toxicokinetics study during 120 h. Cmax (nmol/ml (g.pro))
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
Cmin (nmol/ml (g.pro.))
SPAs
UPAS
HPAs
8.6 ± 3.2 13.3 ± 1.2 22.3 ± 1.0 37.6 ± 3.9
51.6 ± 3.4 78.8 ± 1.5 135.2 ± 9.6 258.7 ± 3.0
312.8 378.0 412.2 469.9
SPAs ± ± ± ±
68.4 28.2 32.3 43.4
3.2 4.2 5.1 9.0
± ± ± ±
0.4 0.1 1.1 1.6
UPAS
HPAs
1.5 ± 0.2 7.0 ± 0.3 12.5 ± 3.2 21.7 ± 0.4
244.4 336.6 362.8 416.4
± ± ± ±
87.8 57.6 52.7 61.1
Table 2a Toxicokinetics during the recovery phase.
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
AUC(0-t)
T1/2(h)
SPAs
UPAS
HPAs
1008.0 ± 134.4 2755.2 ± 588.0 6434.4 ± 856.8 10550.4 ± 957.6
4720.8 ± 456.9 19370.4 ± 7728.0 38220.0 ± 3376.8 78842.4 ± 7543.2
201112.8 429391.2 720232.8 737940.0
SPAs ± ± ± ±
40185.6 48938.4 58531.2 95692.8
100.8 134.4 117.6 151.2
UPAS ± ± ± ±
16.8 15.7 14.9 15.9
151.3 117.9 218.4 453.6
HPAs ± ± ± ±
11.5 16.0 33.6 201.6
3662.4 3679.2 3561.6 5863.2
± ± ± ±
487.2 974.4 957.6 1008.0
Table 2b Toxicokinetics during the recovery phase.
0.5 g/kg 1.0 g/kg 2.0 g/kg 4.0 g/kg
Cmax (nmol/ml (g.pro.))
Cmin (nmol/ml (g.pro.))
SPAs
UPAS
HPAs
4.6 ± 1.2 15.2 ± 5.9 40.8 ± 9.0 48.9 ± 8.2
22.3 ± 2.3 61.6 ± 32.2 136.4 ± 11.6 285.1 ± 67.3
241.6 364.2 513.9 528.4
SPAs ± ± ± ±
28.6 42.1 81.9 96.3
0.7 0.8 0.7 0.9
± ± ± ±
UPAS 0.0 0.1 0.2 0.1
1.1 0.9 0.8 1.9
± ± ± ±
HPAs 0.2 0.2 0.2 0.5
158.4 253.8 276.2 254.5
± ± ± ±
30.6 43.4 22.6 18.4
s, and 17.6 ± 5.1 m/s, which were 53.7 %, 55.2 %, and 39.2 % that of the control animals (43.7 ± 12.0 m/s), respectively. However, rats in the 0.5 g/kg group had an MCV of 41.7 ± 9.8 m/s, showing no difference from the control group (P > 0.05) (Fig. 2G). During the recovery phase, the MCVs of the rats exposed to 1.0, 2.0, and 4.0 g/kg nhexane had increased to the levels of the control animals after 7, 11, and 12 weeks, respectively (Fig. 2H). All MCVs of the impaired rats returned to normal levels by the 31st week (Fig. 2I).
exposed to n-hexane showed a significant increase in accumulation in a dose-dependent manner compared to the control. In the recovery phase, the pyrrole adducts could still be detected in the hair, although their levels had decreased. That is, even after 14 weeks after exposure, the pyrrole adducts were significantly higher in the hair of rats treated with 0.5, 1.0, 2.0, and 4.0 g/kg n-hexane, as compared to that of the control group (P < 0.01) (Fig. 3H).
3.3. Pyrrole adduct measurements
3.4. Toxicokinetic studies
As shown in Fig. 3A, the pyrrole adducts in rat serum of all experimental groups showed significant increases in a dose-dependent manner compared to the control (P < 0.01). In the recovery phase, pyrrole adducts in the sera of rats treated with 0.5, 1.0, 2.0, and 4.0 g/ kg of n-hexane returned to the levels of the control after 1, 2, 4, and 5 weeks of exposure, respectively, which also showed a dose-dependent relationship (Fig. 3B). As shown in Fig. 3D, the pyrrole adducts in the urine of rats in all experimental groups increased in a dose-dependent manner (P < 0.01). Increased levels of pyrrole adducts in the urine of rats treated with 0.5, 1.0, 2.0, and 4.0 g/kg n-hexane were eliminated during the recovery phase after 4, 6, 9 and 12 weeks, respectively (Fig. 3E). As shown in Fig. 3G, pyrrole adducts in the hair of rats
As shown in Fig. 4, serum pyrrole adducts in rats treated with nhexane reached their peak values 12 h after treatment. The half-life of serum pyrrole adducts was 62.9 ± 15.1 h (Table 1a). In addition, the pyrrole adducts in the urine reached their peak values 8–9 h after treatment, with a half-life of 47.8 ± 5.1 h (Table 1a). Interestingly, the concentrations of pyrrole adducts in hair mainly increased by the end of the experiment (Table 1b). The half-lives were then calculated with data collected during the recovery phase, and the results showed that the half-lives of the pyrrole adducts in serum, urine, and hair were 0.8 ± 0.1, 1.4 ± 0.6, and 24.9 ± 7.5 weeks, respectively (Table 2a). The maximum and minimum concentrations of serum, urine and hair pyrrole adducts were shown in Table 2b. 16
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that the rats were exposed to. The rats in the 0.5 g/kg n-hexane dose group showed no behavioral impairments or increase in the pyrrole adduct concentrations in the serum or urine, whereas the pyrrole adducts in hair reached the level of 238.4 ± 24.4 nmol/g protein. (C1). The rats in the 4.0 g/kg dose group showed neuroimpairments starting from the 3rd week, with hair pyrrole adducts reaching 332.7 ± 76.4 nmol/g protein, whereas the decrease in weight gain began in the 2nd week when the hair pyrrole adduct levels were 312.8 ± 98.6 nmol/g protein (C2). Therefore, the NOAEL was set as 275.2 ± 61.5 nmol/g protein (1/2 of (C1+C2)) after two weeks of exposure. 4. Discussion It has been decades since researchers found that n-hexane overexposure could induce peripheral neuropathy in workers. However, the specific treatments for n-hexane intoxication are not yet available, which makes the elucidation of potential preventative strategies very important. In the present study, hair pyrrole adducts were identified as an effective long-term biomarker of n-hexane exposure, and an NOAEL value of n-hexane intoxication was defined based on data from the rat model. These findings show great potential to help prevent occupational intoxication of n-hexane in the future. First, the dose-dependence of the change in weight gain in the rats treated with n-hexane was compared to that of the control animals. The results showed that weight gain of rats treated with n-hexane doses of 1.0, 2.0, and 4.0 g/kg had decreased by the end of the intoxication, whereas the comparison between the 0.5 g/kg dose group and the control showed no significant difference, which was consistent with our previous studies (Yin et al., 2014b). The weights of the affected rats all returned to levels comparable to those of the control animals at the end of the recovery phase. These results revealed that the doses of n-hexane that were used to establish the n-hexane intoxication model in rats were appropriate. To detect the phenotype of peripheral nerve impairment in the nhexane-treated rats, three standard parameters were measured throughout the treatment and recovery phases. The results showed that the onset of gait score changes and decrease in rotarod latency were observed first, followed by decrease in MCV. Moreover, during the recovery phase, it took a longer time for the MCV to be restored in the rats than for the muscles to recover, which was approximately 4 weeks. This time lag might suggest the relatively weak recoverability of the nervous system. The present study is the first to capture the spontaneous rehabilitation of peripheral nerve impairment induced by n-hexane in rats, which is consistent with case reports in humans showing that patients completely recovered after 1–2 years after exposure (Huang, 2008). Thus, these findings recapitulate and verify the phenomenon that n-hexane-induced peripheral neuropathy is reversible. After the establishment of the n-hexane intoxication model, we tried to identify the proper biomarkers for the assessment of the exposure level. 2,5-HD has a relatively short half-life (Yin et al., 2013), is not stable, and easily reacts with other substances to form pyrrole adducts (Torres et al., 2014b). Our previous study had demonstrated that concentrations of pyrrole adducts in the serum and urine of n-hexanetreated rats gradually increased in a dose-dependent manner during the treatment phase (Yin et al., 2014b). Therefore, in the current study, the levels of pyrrole adducts in rat serum, urine, and hair were determined in both the treatment and recovery stages. During the treatment phase, consistent with our previous report, our current results showed that the concentrations of pyrrole adducts in the serum, urine, and hair increased in a dose-dependent manner with n-hexane administration. However, during the recovery phase, the concentrations of pyrrole adducts in the serum and urine drastically dropped and returned to the levels of the control animals at the end of the recovery phase. These results suggest that pyrrole adducts in serum and urine had short half-lives and might not be able to serve as effective biomarkers.
Fig. 5. Correlation analysis. A strong correlation between the concentrations of the pyrrole adducts in serum, urine, and hair and the gait scores of both the treatment and recovery phases.
3.5. Correlation analysis and calculation of NOAEL As shown in Fig. 5, correlation analyses showed that there was a strong correlation between the concentrations of the pyrrole adducts in serum, urine, and hair and the gait scores of both the treatment and recovery phases. In the treatment phase (T), the concentrations of the pyrrole adducts in the serum, urine, and hair all showed strong correlation to the gait scores, with correlation coefficients of 0.701, 0.670, and 0.683, respectively (P < 0.01). A similar correlation was seen between the pyrrole adducts in the serum, urine, and hair of the rats exposed to n-hexane and their gait scores during the recovery phase (R), with correlation coefficients of 0.456, 0.570, and 0.702, respectively (P < 0.01). Moreover, there was strong correlation between the rotarod latency and MCV results and the gait scores in both phases (P < 0.01). In addition, multiple regression analysis was performed in which the behavioral indices were defined as Y, and the concentrations of serum, urine, and hair pyrrole adducts were defined as X (Fig. 6). The results showed that the multiple regression coefficients (multiple R) of the gait scores were the largest among the three indices in both the intoxication and recovery phases (Table 3a). Therefore, the gait score was set as the gold standard for the calculation of the correlation coefficients of each pyrrole adduct. he partial correlation coefficients were also calculated (Table 3b). In the treatment phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.193, 0.120, and 0.324, respectively, and in the recovery phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.050, 0.115, and 0.322, respectively. The accumulation of pyrrole adducts in hair continued with the growth of hair. Thus, we hypothesized that the pyrrole adducts in hair might be able to provide a real-time status of the total level of n-hexane 17
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Fig. 6. Mutiple regression analysis. In the treatment phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.193, 0.120, and 0.324, respectively, and in the recovery phase, the partial correlation coefficients of the serum, urine, and hair pyrrole adducts were 0.050, 0.115, and 0.322, respectively.
Interestingly, in contrast to the serum and urine data, levels of pyrrole adducts in the hair of rats with n-hexane intoxication showed a slower and steady increase during the treatment phase as well as a long-term and sustained presence during the recovery phase. These results suggest that the levels of pyrrole adducts in hair could be a promising biomarker to monitor and diagnose n-hexane intoxication. Occupational monitoring of 2,5-HD set a BEL value at no more than 0.4 mg/l in urine
collected at the end of the workweek. However, as mentioned earlier, the short half-life and instability of 2,5-HD interfere with its monitoring. In contrast, our results showed that hair pyrrole adducts seemed to have longer half-lives and were much more stable. A quick digestion of hair by a combination of NaOH and trypsin, followed by spectrophotometric measurement could determine the pyrrole adduct concentrations. Although spectrophotometry is not as advanced as gas 18
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recovery were within the NOAEL value. Further studies are needed for the definition of the BEL value in humans.
Table 3a Multiple regression analysis results. Variables
PAs T PAs R
Multiple R
P value
Gait score
Rota-rod
MCV
Gait score
Rota-rod
MCV
0.746** 0.587**
−0.499** −0.450*
−0.443** −0.321#
0.000 0.000
0.000 0.003
0.000 0.086
5. Conclusions First, there was a strong positive correlation between the hair pyrrole adduct concentration and peripheral neuropathy, and the hair pyrrole adducts had higher partial correlation coefficients than serum and urine pyrrole adducts in both the treatment and recovery phases. Second, the pyrrole adducts in hair showed remarkable potential to be efficient biomarkers for the prevention and diagnosis of occupational nhexane intoxication because they had much longer excretion half-lives (21–34 weeks) than those of serum and urine. Third, based on our rat model, we suggest that hair pyrrole adduct levels of less than 275.2 ± 61.5 nmol/g protein be referred to as the NOAEL for n-hexane exposure.
*P < 0.05, **P < 0.01, #P > 0.05. Table 3b Correlation analysis ad partial correlation analysis. Variables
SPAs T UPAs T HPAs T SPAs R UPAs R HPAs R
Correlation analysis
Partial correlation analysis
Gait score
P value
Gait score
P value
0.701** 0.669** 0.693** 0.535** 0.601** 0.702**
0.000 0.000 0.000 0.000 0.000 0.000
0.193** 0.120* 0.324** −0.005# 0.241** 0.488**
0.000 0.019 0.000 0.700 0.001 0.000
CRediT authorship contribution statement Xianjie Li: Project administration, Methodology, Formal analysis, Software, Visualization, Writing - original draft. Lulu Jiang: Writing review & editing. Ting Yu: Project administration. Ming Li: Project administration. Qiong Wang: Project administration. Zhidan Liu: Project administration. Keqin Xie: Conceptualization, Funding acquisition, Supervision, Writing - review & editing.
*P < 0.05, **P < 0.01, #P > 0.05.
chromatography, it enables a rapid screening measurement and a longterm surveillance every single week. The toxicokinetic results showed that pyrrole adduct concentrations in serum and urine reached their peak values at 12 h and 8 h, respectively, and then began to fall, whereas the concentrations of the hair pyrrole adducts increased slightly during the five-day test. The halflives of the hair pyrrole adducts could not be calculated during the fiveday test because the hair pyrrole adducts were detectable for a much longer time. Thus, we used the data from the recovery phase to calculate the excretion half-lives of the hair pyrrole adducts when the chronic exposure was discontinued. The excretion half-lives of the pyrrole adducts in the serum and urine were approximately 100–151.2 h (0.7–0.9 week) and 117.6–453.6 h (0.7–2.7 weeks). In contrast, the half-life of the hair pyrrole adducts was almost 3561.6–5947.2 h (21.2–35.4 weeks). Thus, there was a strong positive correlation between the concentrations of the hair pyrrole adducts at the end and n-hexane induced peripheral neuropathy. These results further demonstrate that hair pyrrole adducts have the potential to evaluate the peripheral nerve system status of organisms, regardless of the duration of exposure to n-hexane. In the present study, we analyzed the correlation between the levels of pyrrole adducts in the hair and peripheral neuropathy under different doses of n-hexane intoxication over a long time course. Results of the correlation analysis showed that pyrrole adducts in serum, urine, and hair all showed a strong, positive correlation to the gait scores, which was also suggested by previous studies (Yin et al., 2013; Wang et al., 2017a; Li et al., 2018). Notably, the pyrrole adducts in hair had higher partial correlation coefficients than urine and serum pyrrole adducts. Moreover, hair pyrrole adducts showed the strongest relationship with the onset and disappearance of peripheral nervous system impairments among the three biosamples. Our previous in vitro study also found that there was no difference between the rates of formation of pyrrole adducts in human and rat hair samples after treatment with 2.5-HD; hence the hair pyrrole adducts in workers might also accumulate in a similar manner and indicate the effects of exposure to n-hexane. Therefore, we chose the pyrrole adducts in the hair to establish the NOAEL value. Taking the final concentration of hair pyrrole adducts in rats of the 0.5 g/kg dosage group (238.4 ± 24.4 nmol/g protein) and the minimum concentration influencing the weight gain in the 4.0 g/kg dose group (312.8 ± 98.6 nmol/g.protein), the NOAEL was set as 275.2 ± 61.5 nmol/g protein after two weeks of exposure. Moreover, the levels of hair pyrrole adducts in impaired rats after
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