- Email: [email protected]
PDCB does not promote CNS autoimmunity in the context of genetic susceptibility but worsens its outcome
Journal of Neuroimmunology 323 (2018) 53–55
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short Communication
PDCB does not promote CNS autoimmunity in the context of genetic susceptibility but worsens its outcome☆
T
Divyanshu Dubeya,b,c,1, Rehana Z. Hussaina,1, William A. Miller-Littlea, Amber Salterd, ⁎ Richard Doelgera, Olaf Stüvea,e, a
Department of Neurology & Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, United States Department of Neurology, Mayo Clinic, Rochester, United States c Department of Neurology, Brigham & Women's Hospital and Massachusetts's General Hospital, Boston, United States d Division of Biostatistics, Washington University School of Medicine, St. Louis, United States e Department of Neurology, VA North Texas Health Care System, Medical Service, Dallas, United States b
A B S T R A C T
Background: Para-dichlorobenzene (PDCB) is an aromatic hydrocarbon contained in mothballs that is potentially neurotoxic. A potential pathogenic role of PDCB in MS pathogenesis has been suggested. Methods: To determine the ability of chronic PDCB ingestion to induce CNS autoimmunity in a genetically susceptible mammalian species, naive myelin oligodendrocyte glycoprotein peptide (MOGp)35–55 T cell receptor (TCR) transgenic mice (2D2) on the C57Bl/6 background were orally gavaged once daily with corn oil control, 125 mg/kg PDCB, or 250 mg/kg PDCB for 45 days. The incidence of spontaneous EAE is increased in this mouse strain. Results: Both PDCB treatment groups showed the same spontaneous incidence of EAE, an earlier disease onset, and a slight decrease in survival for 125 mg/kg PDCB mice compared to control mice. We were unable to detect any PDCB, or its metabolites 2,5-dichlorophenol, 2,5-dicholormethylsulfide, and 2,5-dichloromethylsulfone in the brain and spinal cord of control mice. In contrast, PDCB was readily detectable in both compartments in mice who received PDCB via oral gavage, with concentrations being significantly higher in the brain (p < 0.01). Levels of the metabolites 2,5-dichlorophenol and 2,5-dichloromethylsulfone were also significantly higher in brains compared to spinal cords. Conclusion: Our study refutes the hypothesis that PDCB or its metabolites trigger spontaneous T cell-mediated CNS autoimmunity in the setting of genetic susceptibility. A slight increase in mortality with PDCB exposure may be due systemic toxicity of hydrocarbons.
1. Introduction In this journal, we recently reported the case of a 38 years-old female patient with relapsing-remitting multiple sclerosis (RRMS) who had ingested mothballs for recreational purposes (Hession et al., 2014). Despite years of treatment with natalizumab, she continued to accumulate clinical and paraclinical disease activity rapidly. The patient was subsequently found to have had ingested mothballs for recreational purposes. Multiple sclerosis (MS) is inherited as a complex multifactorial disorder with an assumed autoimmune pathogenesis that results from the interaction of genetic and environmental factors. Likely, numerous environmental factors are required to brake immune tolerance in genetically susceptible individuals. Para-dichlorobenzene (PDCB) is an
aromatic hydrocarbon that is potentially neurotoxic. PDCB is an almost ubiquitous environmental contaminant that is absorbed orally, via inhalation, or through dermal exposure. Accumulation of PDCB and its metabolites in sediments and food crops was demonstrated to subsequently contaminate aquatic species used for food consumption in humans (http://www.cdpr.ca.gov/docs/emon/pubs/fatememo/paradichl orobenzene.pdf). Population-based studies detected elevated PDCB tissue levels in a high percentage of individuals without a history of intentional ingestions. A potential pathogenic role of PDCB in MS pathogenesis is intriguing because it is highly lipophilic nature and its propensity to accumulate in lymphatic fluid and in the CNS (Dubey et al., 2014). PDCB is known to cause multi-organ toxicity, including toxicity of the nervous system. Its toxic effects on a cellular and molecular level within the CNS
☆ Olaf Stüve: Dr. Olaf Stüve serves on the editorial boards of the Multiple Sclerosis Journal, and Therapeutic Advances in Neurological Disorders. He has served on data monitoring committees for Pfizer and TG Therapeutics without monetary compensation. Dr. Stüve has advised EMD Serono and Genzyme. He currently receives grant support from Sanofi Genzyme. Dr. Stüve received travel support from Shire. ⁎ Corresponding author at: Department of Neurology & Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, United States. E-mail address: [email protected] (O. Stüve). 1 These authors contributed equally.
https://doi.org/10.1016/j.jneuroim.2018.07.012 Received 22 June 2018; Received in revised form 24 July 2018; Accepted 24 July 2018 0165-5728/ © 2018 Published by Elsevier B.V.
Journal of Neuroimmunology 323 (2018) 53–55
D. Dubey et al.
have not been studied. Clinical manifestations associated with PDCB exposure include cognitive decline, memory deficits, psychomotor retardation, balance impairment, speech problems, vision loss, stupor and coma. As PDCB toxicity can affect any part of nervous system, there are no signs or symptoms specific for PDCB-induced neurotoxicity. Molecular pathways by which the accumulation of PDCB in lymphoid organs may impact the activation and differentiation of lymphocytes and contribute to autoimmunity has never been studied. In the current study, we evaluated the potential role of PDCB and its metabolites in triggering and perpetuating central nervous system (CNS) autoimmunity in the setting of genetic susceptibility. 2. Methods 2.1. Experimental autoimmune encephalomyelitis To determine the ability of chronic PDCB ingestion to induce CNS autoimmunity in a genetically susceptible mammalian species, groups of five female naive myelin oligodendrocyte glycoprotein peptide (MOGp)35–55 T cell receptor (TCR) transgenic mice (2D2) on the C57Bl/6 background were orally gavaged once daily with corn oil control, 125 mg/kg PDCB, or 250 mg/kg/BW PDCB for 45 days (Suhua et al., 2010). The incidence of spontaneous EAE is increased in these animals. Clinical disease incidence and severity was scored daily for 88 days. 2.2. Toxicology To verify and quantify accumulation of PDCB and its metabolites within in the CNS, brains and spinal cords were resected at day 30, and gas chromatography–mass spectrometry (GCMS) was performed at the Small Molecule Mass Spectrometry Facility at Harvard University. 2.3. Flow cytometry Figure 1. Para-dichlorobenzene (PDCB) does not affect disease in genetically susceptible animals. (A) Cumulative probability of spontaneous experimental autoimmune encephalomyelitis (EAE), (B) (EAE) disease course, and (C) probability of death during EAE in 2D2 mice orally gavaged once daily with corn oil (Ctrl), PDCB 125 mg/kg, and 250 mg/kg). There was a trend towards an earlier disease onset and a higher mortality rate in both PDCB treatment groups. Pooled data from two experiments with 5 mice in each treatment group are shown.
Leukocytes were isolated from brain and spinal cord at day 30 (+/− 1 day) (Hussain et al., 2018), and lymphocyte and myeloid subsets were immunophenotyped by multi-parameter flow cytometry (Hussain et al., 2018). Data were acquired using a BD FACS LSR Fortessa, and were analyzed using FlowJo v10 (Becton Dickinson, Franklin Lakes, NJ). 2.4. Statistical analysis
dichloromethylsulfone in the brain and spinal cord of control mice. In contrast, PDCB was readily detectable in both compartments in mice who had been administered PDCB via oral gavage, with concentrations being significantly higher in the brain (p < 0.01) (Fig. 2A). The metabolites 2,5-dichlorophenol and 2,5-dichloromethylsulfone were also significantly higher expressed in the brain compared to the spinal cord in PDCB-fed mice (data not shown). PDCB and its metabolites do not alter the composition of immune cells in the CNS. There was no significant difference among lymphocyte subsets obtained from brains (Fig. 2B) and spinal cords (Fig. 2C) of mice gavaged with PDCB or control mice.
Normally distributed values were compared using the unpaired twosided Student t-test. Differences between two or more means were tested by Analysis of Variance (ANOVA). Survival analysis used the Kaplan Meier method and the log-rank test was used to evaluate differences between groups. A p < 0.05 indicated statistical significance. Analyses were performed with Prism 7 (Graphpad, La Jolla, CA) and SAS v9.4. All experiments were repeated at least once. 3. Results 3.1. PDCB and its metabolites do not change EAE incidence
4. Discussion
Both PDCB treatment groups showed the same spontaneous incidence of EAE (Fig. 1A), an earlier disease onset (Fig. 1B), and a slight decrease in survival for 125 mg/kg PDCB mice (Fig. 1C) compared to control mice.
Our study refutes the hypothesis that PDCB or its metabolites trigger spontaneous CNS autoimmunity in the setting of genetic susceptibility. Elevated concentrations of PDCB and its metabolites in brain and spinal cord provide evidence of their ability to cross the blood-brain-barrier and accumulate in the CNS. However, the absence of a clinical phenotype, or a change in the composition and numbers of immune cells in the CNS of experimental animals suggest that PDCB does not increase the risk of T cell-mediated CNS autoimmunity in this experimental
3.2. Chronic oral ingestion leads to accumulation of PDCB and its metabolites We were unable to detect any PDCB, or its metabolites 2,5-dichlorophenol, 2,5-dicholormethylsulfide, and 2,554
Journal of Neuroimmunology 323 (2018) 53–55
D. Dubey et al.
model. A slight increase in mortality with PDCB exposure may be due to systemic toxicity of hydrocarbons (Hernandez et al., 2010; Weintraub et al., 2000). Limitations of our study include the fact that the full spectrum of PDCB toxicity in the C57BL/6 mouse strain was not investigated. PDCB appears to be neurotoxic in mice with an acute oral LD50 of 533 mg/kg in males and 710 mg/kg in females (Campbell and Davidson, 1970). Our experimental approach was also based on observations by Suhua and colleagues (Suhua et al., 2010), who showed that PDCB at doses of 900 mg/kg/BW are acutely hepatotoxic and nephrotoxic after only 7 days of oral ingestion. In our experiments, we wanted to recapitulate the proposed situation in humans, where chronic low-dose ingestion of PDCB leads to its tissue accumulation and organ toxicity. Potential conflicts of interests Divyanshu Dubey: None. Rehana Z. Hussain: None. William A. Miller-Little: None. Amber Salter: None. Acknowledgments This work was supported by a Pilot grant from the National Multiple Sclerosis Society (NMSS) to OS (PP-1503-03493). References Campbell, D.M., Davidson, R.J., 1970. Toxic haemolytic anaemia in pregnancy due to a pica for paradichlorobenzene. J. Obstet. Gynaecol. British Commonw. 77, 657–659. Dubey, D., Sharma, V.D., Pass, S.E., Sawhney, A., Stuve, O., 2014. Para-dichlorobenzene toxicity - a review of potential neurotoxic manifestations. Ther. Adv. Neurol. Disord. 7, 177–187. Hernandez, S.H., Wiener, S.W., Smith, S.W., 2010. Case files of the new York City poison control center: paradichlorobenzene-induced leukoencephalopathy. J. Med. Toxicol. 6, 217–229. Hession, R.M., Sharma, V., Spiegel, D.E., Tat, C., Hwang, D.G., Dieppa, M., et al., 2014. Multiple sclerosis disease progression and paradichlorobenzene: a tale of mothballs and toilet cleaner. JAMA Neurol. 71, 228–232. http://www.cdpr.ca.gov/docs/emon/ pubs/fatememo/paradichlorobenzene.pdf. Hussain, R.Z., Miller-Little, W.A., Doelger, R., Cutter, G.R., Loof, N., Cravens, P.D., et al., 2018. Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE. Neurol Neuroimmunol. Neuroinflamm. 5, e437. Suhua, W., Rongzhu, L., Changqing, Y., Guangwei, X., Fangan, H., Junjie, J., et al., 2010. Lipid peroxidation and changes of trace elements in mice treated with paradichlorobenzene. Biol. Trace Elem. Res. 136, 320–336. Weintraub, E., Gandhi, D., Robinson, C., 2000. Medical complications due to mothball abuse. South. Med. J. 93, 427–429.
Fig. 2. Para-dichlorobenzene (PDCB) accumulates in fatty tissues, but does not affect the composition or number of leukocytes in the central nervous system (CNS). (A) PDCB was readily detectable by gas chromatography–mass spectrometry in brain and spinal cords of mice who had been administered PDCB via oral gavage, with concentrations being significantly higher in the brain. Immunophenotyping by multi-parameter flow cytometry of leukocytes isolated from (B) brains and (C) spinal cords of mice orally gavaged once daily with corn oil (Ctrl) or PDCB 250 mg/kg showed no significant difference between the two groups.
55
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short Communication
PDCB does not promote CNS autoimmunity in the context of genetic susceptibility but worsens its outcome☆
T
Divyanshu Dubeya,b,c,1, Rehana Z. Hussaina,1, William A. Miller-Littlea, Amber Salterd, ⁎ Richard Doelgera, Olaf Stüvea,e, a
Department of Neurology & Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, United States Department of Neurology, Mayo Clinic, Rochester, United States c Department of Neurology, Brigham & Women's Hospital and Massachusetts's General Hospital, Boston, United States d Division of Biostatistics, Washington University School of Medicine, St. Louis, United States e Department of Neurology, VA North Texas Health Care System, Medical Service, Dallas, United States b
A B S T R A C T
Background: Para-dichlorobenzene (PDCB) is an aromatic hydrocarbon contained in mothballs that is potentially neurotoxic. A potential pathogenic role of PDCB in MS pathogenesis has been suggested. Methods: To determine the ability of chronic PDCB ingestion to induce CNS autoimmunity in a genetically susceptible mammalian species, naive myelin oligodendrocyte glycoprotein peptide (MOGp)35–55 T cell receptor (TCR) transgenic mice (2D2) on the C57Bl/6 background were orally gavaged once daily with corn oil control, 125 mg/kg PDCB, or 250 mg/kg PDCB for 45 days. The incidence of spontaneous EAE is increased in this mouse strain. Results: Both PDCB treatment groups showed the same spontaneous incidence of EAE, an earlier disease onset, and a slight decrease in survival for 125 mg/kg PDCB mice compared to control mice. We were unable to detect any PDCB, or its metabolites 2,5-dichlorophenol, 2,5-dicholormethylsulfide, and 2,5-dichloromethylsulfone in the brain and spinal cord of control mice. In contrast, PDCB was readily detectable in both compartments in mice who received PDCB via oral gavage, with concentrations being significantly higher in the brain (p < 0.01). Levels of the metabolites 2,5-dichlorophenol and 2,5-dichloromethylsulfone were also significantly higher in brains compared to spinal cords. Conclusion: Our study refutes the hypothesis that PDCB or its metabolites trigger spontaneous T cell-mediated CNS autoimmunity in the setting of genetic susceptibility. A slight increase in mortality with PDCB exposure may be due systemic toxicity of hydrocarbons.
1. Introduction In this journal, we recently reported the case of a 38 years-old female patient with relapsing-remitting multiple sclerosis (RRMS) who had ingested mothballs for recreational purposes (Hession et al., 2014). Despite years of treatment with natalizumab, she continued to accumulate clinical and paraclinical disease activity rapidly. The patient was subsequently found to have had ingested mothballs for recreational purposes. Multiple sclerosis (MS) is inherited as a complex multifactorial disorder with an assumed autoimmune pathogenesis that results from the interaction of genetic and environmental factors. Likely, numerous environmental factors are required to brake immune tolerance in genetically susceptible individuals. Para-dichlorobenzene (PDCB) is an
aromatic hydrocarbon that is potentially neurotoxic. PDCB is an almost ubiquitous environmental contaminant that is absorbed orally, via inhalation, or through dermal exposure. Accumulation of PDCB and its metabolites in sediments and food crops was demonstrated to subsequently contaminate aquatic species used for food consumption in humans (http://www.cdpr.ca.gov/docs/emon/pubs/fatememo/paradichl orobenzene.pdf). Population-based studies detected elevated PDCB tissue levels in a high percentage of individuals without a history of intentional ingestions. A potential pathogenic role of PDCB in MS pathogenesis is intriguing because it is highly lipophilic nature and its propensity to accumulate in lymphatic fluid and in the CNS (Dubey et al., 2014). PDCB is known to cause multi-organ toxicity, including toxicity of the nervous system. Its toxic effects on a cellular and molecular level within the CNS
☆ Olaf Stüve: Dr. Olaf Stüve serves on the editorial boards of the Multiple Sclerosis Journal, and Therapeutic Advances in Neurological Disorders. He has served on data monitoring committees for Pfizer and TG Therapeutics without monetary compensation. Dr. Stüve has advised EMD Serono and Genzyme. He currently receives grant support from Sanofi Genzyme. Dr. Stüve received travel support from Shire. ⁎ Corresponding author at: Department of Neurology & Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, United States. E-mail address: [email protected] (O. Stüve). 1 These authors contributed equally.
https://doi.org/10.1016/j.jneuroim.2018.07.012 Received 22 June 2018; Received in revised form 24 July 2018; Accepted 24 July 2018 0165-5728/ © 2018 Published by Elsevier B.V.
Journal of Neuroimmunology 323 (2018) 53–55
D. Dubey et al.
have not been studied. Clinical manifestations associated with PDCB exposure include cognitive decline, memory deficits, psychomotor retardation, balance impairment, speech problems, vision loss, stupor and coma. As PDCB toxicity can affect any part of nervous system, there are no signs or symptoms specific for PDCB-induced neurotoxicity. Molecular pathways by which the accumulation of PDCB in lymphoid organs may impact the activation and differentiation of lymphocytes and contribute to autoimmunity has never been studied. In the current study, we evaluated the potential role of PDCB and its metabolites in triggering and perpetuating central nervous system (CNS) autoimmunity in the setting of genetic susceptibility. 2. Methods 2.1. Experimental autoimmune encephalomyelitis To determine the ability of chronic PDCB ingestion to induce CNS autoimmunity in a genetically susceptible mammalian species, groups of five female naive myelin oligodendrocyte glycoprotein peptide (MOGp)35–55 T cell receptor (TCR) transgenic mice (2D2) on the C57Bl/6 background were orally gavaged once daily with corn oil control, 125 mg/kg PDCB, or 250 mg/kg/BW PDCB for 45 days (Suhua et al., 2010). The incidence of spontaneous EAE is increased in these animals. Clinical disease incidence and severity was scored daily for 88 days. 2.2. Toxicology To verify and quantify accumulation of PDCB and its metabolites within in the CNS, brains and spinal cords were resected at day 30, and gas chromatography–mass spectrometry (GCMS) was performed at the Small Molecule Mass Spectrometry Facility at Harvard University. 2.3. Flow cytometry Figure 1. Para-dichlorobenzene (PDCB) does not affect disease in genetically susceptible animals. (A) Cumulative probability of spontaneous experimental autoimmune encephalomyelitis (EAE), (B) (EAE) disease course, and (C) probability of death during EAE in 2D2 mice orally gavaged once daily with corn oil (Ctrl), PDCB 125 mg/kg, and 250 mg/kg). There was a trend towards an earlier disease onset and a higher mortality rate in both PDCB treatment groups. Pooled data from two experiments with 5 mice in each treatment group are shown.
Leukocytes were isolated from brain and spinal cord at day 30 (+/− 1 day) (Hussain et al., 2018), and lymphocyte and myeloid subsets were immunophenotyped by multi-parameter flow cytometry (Hussain et al., 2018). Data were acquired using a BD FACS LSR Fortessa, and were analyzed using FlowJo v10 (Becton Dickinson, Franklin Lakes, NJ). 2.4. Statistical analysis
dichloromethylsulfone in the brain and spinal cord of control mice. In contrast, PDCB was readily detectable in both compartments in mice who had been administered PDCB via oral gavage, with concentrations being significantly higher in the brain (p < 0.01) (Fig. 2A). The metabolites 2,5-dichlorophenol and 2,5-dichloromethylsulfone were also significantly higher expressed in the brain compared to the spinal cord in PDCB-fed mice (data not shown). PDCB and its metabolites do not alter the composition of immune cells in the CNS. There was no significant difference among lymphocyte subsets obtained from brains (Fig. 2B) and spinal cords (Fig. 2C) of mice gavaged with PDCB or control mice.
Normally distributed values were compared using the unpaired twosided Student t-test. Differences between two or more means were tested by Analysis of Variance (ANOVA). Survival analysis used the Kaplan Meier method and the log-rank test was used to evaluate differences between groups. A p < 0.05 indicated statistical significance. Analyses were performed with Prism 7 (Graphpad, La Jolla, CA) and SAS v9.4. All experiments were repeated at least once. 3. Results 3.1. PDCB and its metabolites do not change EAE incidence
4. Discussion
Both PDCB treatment groups showed the same spontaneous incidence of EAE (Fig. 1A), an earlier disease onset (Fig. 1B), and a slight decrease in survival for 125 mg/kg PDCB mice (Fig. 1C) compared to control mice.
Our study refutes the hypothesis that PDCB or its metabolites trigger spontaneous CNS autoimmunity in the setting of genetic susceptibility. Elevated concentrations of PDCB and its metabolites in brain and spinal cord provide evidence of their ability to cross the blood-brain-barrier and accumulate in the CNS. However, the absence of a clinical phenotype, or a change in the composition and numbers of immune cells in the CNS of experimental animals suggest that PDCB does not increase the risk of T cell-mediated CNS autoimmunity in this experimental
3.2. Chronic oral ingestion leads to accumulation of PDCB and its metabolites We were unable to detect any PDCB, or its metabolites 2,5-dichlorophenol, 2,5-dicholormethylsulfide, and 2,554
Journal of Neuroimmunology 323 (2018) 53–55
D. Dubey et al.
model. A slight increase in mortality with PDCB exposure may be due to systemic toxicity of hydrocarbons (Hernandez et al., 2010; Weintraub et al., 2000). Limitations of our study include the fact that the full spectrum of PDCB toxicity in the C57BL/6 mouse strain was not investigated. PDCB appears to be neurotoxic in mice with an acute oral LD50 of 533 mg/kg in males and 710 mg/kg in females (Campbell and Davidson, 1970). Our experimental approach was also based on observations by Suhua and colleagues (Suhua et al., 2010), who showed that PDCB at doses of 900 mg/kg/BW are acutely hepatotoxic and nephrotoxic after only 7 days of oral ingestion. In our experiments, we wanted to recapitulate the proposed situation in humans, where chronic low-dose ingestion of PDCB leads to its tissue accumulation and organ toxicity. Potential conflicts of interests Divyanshu Dubey: None. Rehana Z. Hussain: None. William A. Miller-Little: None. Amber Salter: None. Acknowledgments This work was supported by a Pilot grant from the National Multiple Sclerosis Society (NMSS) to OS (PP-1503-03493). References Campbell, D.M., Davidson, R.J., 1970. Toxic haemolytic anaemia in pregnancy due to a pica for paradichlorobenzene. J. Obstet. Gynaecol. British Commonw. 77, 657–659. Dubey, D., Sharma, V.D., Pass, S.E., Sawhney, A., Stuve, O., 2014. Para-dichlorobenzene toxicity - a review of potential neurotoxic manifestations. Ther. Adv. Neurol. Disord. 7, 177–187. Hernandez, S.H., Wiener, S.W., Smith, S.W., 2010. Case files of the new York City poison control center: paradichlorobenzene-induced leukoencephalopathy. J. Med. Toxicol. 6, 217–229. Hession, R.M., Sharma, V., Spiegel, D.E., Tat, C., Hwang, D.G., Dieppa, M., et al., 2014. Multiple sclerosis disease progression and paradichlorobenzene: a tale of mothballs and toilet cleaner. JAMA Neurol. 71, 228–232. http://www.cdpr.ca.gov/docs/emon/ pubs/fatememo/paradichlorobenzene.pdf. Hussain, R.Z., Miller-Little, W.A., Doelger, R., Cutter, G.R., Loof, N., Cravens, P.D., et al., 2018. Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE. Neurol Neuroimmunol. Neuroinflamm. 5, e437. Suhua, W., Rongzhu, L., Changqing, Y., Guangwei, X., Fangan, H., Junjie, J., et al., 2010. Lipid peroxidation and changes of trace elements in mice treated with paradichlorobenzene. Biol. Trace Elem. Res. 136, 320–336. Weintraub, E., Gandhi, D., Robinson, C., 2000. Medical complications due to mothball abuse. South. Med. J. 93, 427–429.
Fig. 2. Para-dichlorobenzene (PDCB) accumulates in fatty tissues, but does not affect the composition or number of leukocytes in the central nervous system (CNS). (A) PDCB was readily detectable by gas chromatography–mass spectrometry in brain and spinal cords of mice who had been administered PDCB via oral gavage, with concentrations being significantly higher in the brain. Immunophenotyping by multi-parameter flow cytometry of leukocytes isolated from (B) brains and (C) spinal cords of mice orally gavaged once daily with corn oil (Ctrl) or PDCB 250 mg/kg showed no significant difference between the two groups.
55