Neurotoxicology and Teratology 47 (2015) 89–95

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Abnormal white matter integrity and impairment of cognitive abilities in adolescent inhalant abusers Zeki Yuncu a, Nabi Zorlu b,⁎, Hozan Saatcioglu a, Burge Basay c, Omer Basay c, Pelin Kurtgoz Zorlu d, Omer Kitis e, Fazil Gelal f a

Department of Child Psychiatry, Ege University School of Medicine, Izmir 35040, Turkey Department of Psychiatry, Katip Celebi University Ataturk Training and Research Hospital, Izmir 35360, Turkey Department of Child Psychiatry, Woman Birth and Child Hospital, Aydin 09000, Turkey d Department of Psychiatry, Bozyaka Training and Research Hospital, Izmir 35170, Turkey e Department of Radiodiagnostics, Ege University School of Medicine, Izmir 35040, Turkey f Department of Radiodiagnostics, Katip Celebi University Ataturk Training and Research Hospital, Izmir 35360, Turkey b c

a r t i c l e

i n f o

Article history: Received 26 May 2014 Received in revised form 24 November 2014 Accepted 24 November 2014 Available online 3 December 2014 Keywords: Inhalant abuse Diffusion tensor imaging Tract-based spatial statistics White matter Neuropsychology

a b s t r a c t Inhalant abuse represents a major health problem especially among adolescents and young adults. However, less is known about white matter (WM) microstructure in adolescent inhalant abusers. In the present study, we used diffusion tensor imaging (DTI) to study WM changes in adolescent inhalant abusers compared with healthy controls. We also tested whether there was any relationship between WM integrity and neuropsychological measures in adolescent inhalant abusers. The study included 19 adolescent inhalant abusers and 19 healthy control subjects. Whole brain analysis of WM microstructure was performed using tract-based spatial statistics (TBSS) to detect abnormal WM regions between groups. Wisconsin card sorting test (WCST) and Stroop test were used to measure neuropsychological performance. We found that adolescent inhalant abuser group had significantly higher axial diffusivity (AD) values in left parietal, occipital and temporal WM than in healthy control group. Inhalant abuser and control groups did not differ significantly on fractional anisotropy (FA) and radial diffusivity (RD) values. Adolescent inhalant abusers showed worse performance when compared with control group in WCST and Stroop test. There was no significant correlation of AD values in significant clusters with neuropsychological test performances within the two groups. We only found discrete impairments in neuropsychological test performance and WM integrity in adolescent inhalant abusers compared with healthy control subjects and we were not able to demonstrate a direct correlation between WM alterations and neurocognitive performance. Future work is required to longitudinally evaluate brain abnormalities through methods assessing brain structure, function and connectivity. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Inhalant abuse represents a major health problem especially among adolescents and young adults. The low cost and easy availability of organic solvents have led to increases in the number of young abusers in many countries. Toluene is the major component of organic industrial solvents that is thought to cause neurotoxicity seen in solvent abusers (Filley et al., 2004). Inhalant use typically begins in preadolescence and can continue well into adulthood (Wu and Ringwalt, 2006). Such use may negatively impact brain development, especially white matter (WM) connectivity, which continues to develop throughout adolescence (Lubman et al., 2007; Yücel et al., 2008). Studies examining the

⁎ Corresponding author at: Katip Celebi University Ataturk Training and Research Hospital, Department of Psychiatry, İzmir 35360, Turkey. Tel.: +90 232 2444444x1581. E-mail address: [email protected] (N. Zorlu).

http://dx.doi.org/10.1016/j.ntt.2014.11.009 0892-0362/© 2014 Elsevier Inc. All rights reserved.

effects of inhalant abuse on the brain reported predominantly WM lesions among chronic inhalant abusers (Rosenberg et al., 2002; Aydin et al., 2002; Yücel et al., 2010), possibly due to highly lipophilic nature of toluene that makes it particularly toxic to fatty tissues such as myelin. Diffusion tensor imaging (DTI) is a magnetic resonance imaging technique that is suitable to investigate WM axonal integrity in vivo quantitatively. Fractional anisotropy (FA) is a measure of the degree to which water diffusion is constrained in the brain and is widely used as a general index of axonal integrity (Kubicki et al., 2002). Damage to WM or demyelination along neuronal axons results in more isotropic water movement and is manifested as relatively low FA values. A problem with DTI is the interpretation of changes of FA. Therefore, the component measures from which FA is derived, the so-called first (λ1) and second (λ2, λ3) eigenvalues measuring axial- and radial-diffusions (AD and RD, respectively) to the primary axis of the axon, can provide additional insights regarding the nature of WM deficits. An increase in RD values is thought to signify increased space between fibers

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suggesting demyelination or dysmyelination (Harsan et al., 2006), whereas a decrease in AD values suggests axonal injury (Lazar et al., 2003). Studies examining WM in substance abusing adolescents offer equivocal findings. In adolescent substance abusers, both reductions (Bava et al., 2009; Jacobus et al., 2009; McQueeny et al., 2009) and increases (De Bellis et al., 2008; Bava et al., 2009; Cardenas et al., 2013) in FA have been observed. Differences in AD were also inconsistent between studies, with AD reductions (Ashtari et al., 2009; Thatcher et al., 2010) and increases (Bava et al., 2013) found in participants with substance abuse. To our knowledge, only one DTI study examined whole brain WM microstructure in the adolescent inhalant abusers. Yücel et al. (2010) studied 11 adolescents (mean age 18.2) who used inhalants and 8 matched controls (mean age 19.7) with no history of drug use and found that adolescent inhalant abusers had lower FA in the left hippocampus and in the left and right limbs of the splenium of the corpus callosum than in controls. However, in that study, first, the inhalant abuser group also abused other substances, in particular cannabis; therefore it is difficult to examine how these substances may interact to affect WM integrity; and second, the sample size was small which increases the likelihood of type II error. While inhalants are often among the first drugs used by adolescents (Medina-Mora and Real, 2008), the majority of inhalant research has been performed in adult populations and there has been limited research investigating the effects of inhalant use on executive functions during this key phase of development (Lubman et al., 2008). The findings of the studies that examined neuropsychological functioning in adolescent inhalant abusers are controversial. Some studies indicated that toluene did not lead to neuropsychological impairment (Chadwick et al., 1989; Takagi et al., 2011a); however, other neuropsychological investigations have found significant impairments across many domains of cognitive functioning, including learning and memory, visuospatial reasoning, working memory, and executive abilities (Allison and Jerrom, 1984; Vilar-López et al., 2013; Takagi et al., 2011b). Inconsistent results may be due to differences in methodology and subject characteristics. Inhalant users are characterized by numerous confounds (e.g., psychopathology, comorbid drug use, parental neglect and abuse) that may affect neuropsychological performance, and it is important to consider such factors when examining drug-specific cognitive impairments (Lubman et al., 2008). Executive functioning is a complex construct which involves different cognitive domains. Cognitive deficits are predictors of poor prognosis in substance dependent patients (Turner et al., 2009). Hence it is of great importance to assess neuropsychological functioning, executive functioning in particular. There is no established consensus on which tests are considered the most sensitive in assessing executive functions. In this study, we used Wisconsin Card Sorting Task (WCST) as a measure of cognitive flexibility, which requires flexible adjustment of actions following performance feedback (Stuss and Levine, 2002), and Stroop test as a measure of response inhibition and attentional control (MacLeod, 1992). In the current study, we examined executive functions and brain DTI in a group of adolescent inhalant abusers. We also tested whether there was any relationship between WM integrity and executive functions. We applied Tract Based Spatial Statistics (TBSS), to study WM changes in the adolescent inhalant abusers compared with healthy controls. The skeleton-based approach of TBSS may overcome potential problems with the registration and alignment of WM between subjects in order to allow cross-subject statistical analysis (Smith et al., 2006). 2. Materials and methods 2.1. Subjects We examined adolescents who regularly abused inhalants daily or almost daily for a period of at least 6 months. Inhalant abusers were recruited among adolescents who were admitted to an adolescent

addiction treatment center due to inhalant abuse. The common solvent abused by all the participants was paint thinner and the typical administration way was huffing. Exclusion criteria for adolescent inhalant abusers were as follows: history of any serious psychiatric illness, including any psychotic disorder, bipolar disorder, or major depressive disorder or use of psychoactive medications; history of traumatic brain injury with loss of consciousness exceeding 10 minutes; presence of diseases that may affect the CNS (e.g., meningitis, epilepsy, HIV); history of chronic medical illness; left-handedness; visual impairment, colorblindness or hearing impairments; and contraindications for MRI. In addition, subjects were excluded if they reported use of alcohol more than 10 drinks per week, more than 15 lifetime use of any category of illicit drugs or had a positive urine screen for any illicit drugs. Control subjects met the same criteria with patients, except for the history of substance use disorder. Due to very high incidence of comorbid tobacco use in inhalant abusers, subjects who used tobacco were not excluded. Adolescent inhalant users were abstinent from inhalants at least 5 days (range 5–9) before the day of brain imaging. Sixty-two adolescent inhalant users were invited to participate in the DTI study. Fifty-seven of them agreed and attended the study. Thirty-four individuals were excluded due to either a history of traumatic brain injury with loss of consciousness exceeding 10 minutes (n = 6), diagnosis of exclusionary DSM-IV Axis I disorders (n = 12; i.e., psychotic disorder, major depressive disorder), use of psychoactive medications (n = 19), or regular cannabis use (n = 25). Remaining 23 individuals were examined with MRI at most 5 days later using volatiles. Data of 4 participants were discarded due to technical problems in the DTI sequence, resulting in a total of 19 participants for all subsequent analyses. Nineteen right-handed healthy control subjects were recruited from the community. The information about age of onset for inhalant use and total duration of inhalant use were obtained from the abusers and their parents with a structured interview. Measurement of the exact daily dosage of inhaled toluene was impossible because of the easy vaporization of thinners. In addition, issues related to the methods of abuse such as variability in the frequency of huffing, the amount of thinner inhaled during each huffing episode, and different physical properties of the rags used profoundly affect the reliability of measurements. Therefore, we used the amount of thinner taken weekly (as cans per week) to represent the amount of weekly toluene consumption. Cognitive functions were evaluated by using the Wechsler Intelligence Scale for Children—Revised (WISC-R), Stroop test and WCST within the 2 days of the MRI examination. The project was approved by the Ethics Committee of the Ege University Medical School. Informed written consent was obtained from all participants and their parents prior to testing. 2.2. Neuropsychological assessments 2.2.1. Wechsler Intelligence Scale For Children—Revised (WISC-R) (Wechsler, 1974) WISC-R was used to assess IQ both in the adolescent inhalant abusers and in healthy controls. The WISC-R was adapted and standardized in Turkey in 1997 (Savaşır and Şahin, 1997). 2.2.2. Wisconsin card sorting test (WCST) (Heaton, 1981) WCST was one of the tests used to evaluate frontal cortex functions and considered particularly sensitive to dorsolateral prefrontal cortex functions (Buchsbaum et al., 2005). Computer version of WCST was used in the study. Success depends on the comprehension of matching principle in WCST. One hundred and twenty-eight geometrically designed cards grouped into three in terms of color, form or number were used in the test. There is no time limit. The WCST was adapted and standardized in Turkey in 1998 (Karakaş et al., 1998). Number of

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perseverative errors and number of categories completed were used in the analysis. 2.2.3. Stroop test The present study used the Stroop test TBAG (Turkish Scientific and Technical Research Council of Turkey) version (Karakaş et al., 1998). Stroop test TBAG version represents a combination of the original Stroop test (Stroop, 1935) and the Victoria Version (Spreen and Strauss, 1991). Stroop TBAG has five subtests that sequentially involve reading color words that are printed in black (STR/1), reading colored words that denote different colors (STR/2), naming color of colored circles (STR/ 3), naming color of colored neutral words (STR/4), and naming color of colored words where color and meaning are incongruent for some of the words (STR/5). The test renders scores on time to completion/duration (D), number of errors (E) for each part of the test (1–5). In particular, shorter reaction times in part 5 reflect better cognitive performance such as response inhibition and attention.

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were corrected for family wise error at p b 0.05 and clusters greater than 200 voxels were reported. The most probable anatomic localization of each significant cluster was determined using publicly accessible white matter atlases (http://www.dtiatlas.org). Age and IQ scores were checked for normality of their distribution using Kolmogorov–Smirnov normality test. All variables were distributed normally and independent samples t tests were performed to assess group differences. Chi-square tests were used to compare groups for gender and smoking status. Multiple analysis of covariance (MANCOVA) test was used to examine the group effect (inhalant users or control) on WCST and Stroop scores while controlling for IQ and smoking status. Spearman's correlation analysis was used to explore the associations between TBSS results, neuropsychological test performances, and inhalant use variables because of the small sample size. For all analyses, the p-value was set to b 0.05. Statistical analysis was performed using SPSS version 16. 3. Results

2.3. Imaging protocol 3.1. Demographic, inhalant use and clinical characteristics MR imaging was performed on a 3.0 Tesla scanner (Siemens Magnetom Verio, Numaris/4, Syngo MR B17, Erlangen, Germany) with an 12-channel head matrix coil. DTI data were acquired with a spinecho single-shot, echo-planar imaging sequence (TR/TE: 10600/95 msec, number of diffusion directions: 64, b values: 0 and 1000 s/mm2, GRAPPA factor: 2, voxel size: 2 × 2 × 2 mm, FOV: 256 × 256, matrix: 128 × 128, and no gap).

Table 1 shows the demographic, inhalant use and clinical characteristics of participants. There were significantly more smokers in the adolescent inhalant abusers group than in the control group. The controls had significantly higher IQ scores than in the adolescent inhalant abusers 3.2. TBSS analyses

2.4. DTI data analysis DTI data were analyzed using FMRIB's (Oxford Centre for Functional MRI of the Brain) Diffusion Toolbox, which is part of FSL (FMRIB Software Library) (Smith et al., 2004). Motion and eddy current artifacts were corrected using FSL EDDY_ CORRECT. A brain mask of the nondiffusion weighted image was created using FSL's Brain Extraction Tool (Smith, 2002). The diffusion tensor was then calculated with FSL DTIFIT for whole brain volumes and the resulting FA maps together with the AD (λ1) and RD ((λ2 + λ3)/2) maps, were used in subsequent TBSS analysis. Voxel-wise statistical analysis of the data was performed by using TBSS (Smith et al., 2006). FA data were aligned into a common space using a non-linear registration algorithm (FSL FNIRT) to register the images to the standard space Montreal Neurological Institute 152 template and upsampled to 1 × 1 × 1 mm3. Thereafter, the registered FA images were averaged to generate a cross-subject mean FA image, and then the mean FA image was applied to create a mean FA skeleton which represents the main fiber tracks and the center of all fiber tracts common to the group. The skeleton was then thresholded at an FA value of 0.2 which limits the effects of poor alignment across subjects and reduces the likelihood of inclusion of GM and CSF voxels in the skeleton. The skeleton that was then created contained WM tracts that were common to all subjects. A “distance map” was then created which was used to project each FA image onto the mean FA skeleton that was common to all subjects. AD and RD images were also aligned into MNI space and projected onto the mean FA skeleton using the protocol of non-FA images in TBSS. 2.5. Statistical analyses In order to identify FA, AD and RD differences between inhalant abusing subjects and controls, the skeletonized FA data were fed into the voxel-wise statistical analysis with age, IQ and smoking status as covariates which was based on non-parametric approach utilizing permutation test theory (randomize tool in FSL) (Nichols and Holmes, 2002). The Threshold-Free Cluster Enhancement (TFCE) method was used to define the clusters (Smith and Nichols, 2009). Significant clusters

AD was significantly higher in inhalant abuser group than in the control group in one cluster (Table 2, Fig. 1). Voxels from the AD cluster were found in left occipital, parietal and temporal lobes mainly in the superior longitudinal fasciculus, inferior fronto-occipital fasciculus, inferior longitudinal fasciculus, and in forceps major. Inhalant abusers and controls did not differ significantly for FA and RD values. 3.3. Neuropsychological test performances As shown in Table 3, adolescent inhalant abuser group showed worse performance in WCST and Stroop test than the control group 3.4. Correlation analyses There was no significant correlation between AD values in the significant cluster and neuropsychological test performances within the adolescent inhalant abusers or control group. In addition, there was no significant correlation between AD values in the significant cluster

Table 1 Demographic, inhalant use and clinical characteristics of study participants.

Age (years) % Smokers Sex, % male Verbal IQ Performance IQ Total IQ Alcohol use (standard drinks/week) Duration of inhalant use (months) Age at first use (years) Weekly inhalant use (cans/week)

Adolescent inhalant abusers (n = 19)

Controls (n = 19)

t or χ2

p

15.5 ± 1.0 89.5 84.2 87.4 ± 22.3 93.4 ± 13.0 91.3 ± 15.6 5.3 ± 1.9

16.1 ± 0.8 21.1 78.9 105.3 ± 12.0 99.7 ± 17.9 103.7 ± 12.3 2.6 ± 2.0

t = −2.0 χ2 = 18.0 χ2 = 0.2 t = −3.1 t = −1.2 t = −2.7 t = 4.2

0.06 0.00 0.68 0.00 0.22 0.01 0.00

13.5 ± 8.8 14.4 ± 1.2 12.1 ± 2.2

Data are expressed as mean ± standard deviation.

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Table 2 Mean (± standard deviation) values of AD in the cluster in which higher values were observed in TBSS analysis in adolescent inhalant abusers compared to controls (p b 0.05, TFCE corrected). Cluster regions

Major tracts

Voxels

Adolescent inhalant abusers (n = 19)

Controls (n = 19)

p

X (mm)

Y (mm)

Z (mm)

Parietal Occipital Temporal

L Superior longitidunal fasciculus L Inferior fronto-occipital fasciculus L Inferior longitidunal fasciculus Forceps major

2614

1.36 ± 0.10

1.23 ± 0.18

0.018

121

59

80

X, Y, Z: the location of its peak value in the cluster (MNI coordinates); L = left. AD: ×10−3 mm2/s.

and weekly inhalant use, duration of inhalant use, or age started inhalant use. 4. Discussion In this study, we examined WM integrity throughout the brain and neuropsychological performance in adolescent inhalant abusers compared to healthy controls. We also examined the relationship between WM integrity and neuropsychological performance in adolescent inhalant abusers and healthy controls. We found that adolescent inhalant abuser group had significantly higher AD values in left parietal, occipital and temporal WM than in the healthy control group. Our sample of adolescent inhalant abusers showed worse performance in WCST and Stroop test compared to control group. However, we did not find any significant correlations between AD values in significant cluster and neuropsychological test performances. To date, only one DTI study had assessed WM integrity in adolescent inhalant abusers and thus, our findings are difficult to compare with previous studies. Yücel et al. (2010) found that relative to controls, inhalant abusers had lower FA in the left hippocampus and in the left and right limbs of the splenium of the corpus callosum. Interestingly, our inhalant abuser subjects showed normal FA values, but they did show higher AD values in the left parietal, occipital and temporal lobes in the superior longitudinal fasciculus, inferior fronto-occipital

fasciculus, inferior longitudinal fasciculus and in the forceps major. These different patterns of WM abnormalities might be explained by different sample characteristics in two studies. Yücel et al. (2010) sample was older in age and had a longer duration of inhalant use. In our sample, the mean duration inhalant use was 13.5 months, compared to 3 years reported in previous study (Yücel et al., 2010). Thus, one possible explanation for the lack of FA differences between groups could be that the inhalant group had not used inhalants long enough to develop WM deficits similar to those observed in the previous study. In line with this, Aydin et al. (2002) have found a time dependent effect of inhalant abuse in humans showing that MRI detectable changes were associated with periods of abuse greater than 4 years. Similarly, in rats, Duncan et al. (2012) performed DTI prior to exposure, and after 4 and 8 weeks, to examine the integrity of WM tracts and found that DTI detectable WM abnormalities were only present following 8 weeks of exposure to toluene. Second, adolescent inhalant abusers in our study reported higher rates of cigarette smoking than in the previous study (Yücel et al., 2010). We statistically controlled DTI analysis for cigarette smoking; however contributions of cigarette to deficits in WM integrity remained unclear. Results from a recent study suggest that adolescent smokers may have elevated FA values in several brain regions (Hudkins et al., 2012). Therefore, concurrent nicotine use in the current study could explain the FA differences. Thirdly, in our study inhalant users were excluded if they reported lifetime cannabis use more than

Fig. 1. Cluster (red) in which higher axial diffusivity values were observed in TBSS analysis in adolescent inhalant abusers compared to controls (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Z. Yuncu et al. / Neurotoxicology and Teratology 47 (2015) 89–95 Table 3 Mancova: impact of inhalant use with IQ and smoking status as covariates for WCST and Stroop test. Adolescent inhalant abusers (n = 19) WCST — number of categories completed WCST — number of perseverative errors Stroop test 1/time (s) Stroop test 2/time (s) Stroop test 3/time (s) Stroop test 4/time (s) Stroop test 5/time (s) Stroop test 5/E

Controls (n = 19)

F

df

p

3.5 ± 2.6

6.3 ± 2.0

1.244

1

0.272

31.8 ± 19.3

14.9 ± 5.7

4.296

1

0.046

27.2 ± 4.8 40.2 ± 6.2 30.1 ± 4.2 53.1 ± 11.4 78.7 ± 18.1 3.0 ± 3.0

23.5 ± 2.8 32.3 ± 5.0 25.7 ± 3.0 41.5 ± 8.3 58.1 ± 14.9 2.6 ± 1.9

2.829 9.850 3.138 6.658 6.776 0.064

1 1 1 1 1 1

0.102 0.004 0.085 0.014 0.014 0.802

WCST: Wisconsin card sorting test; E: number of errors; data are expressed as mean (standard deviation).

15 times; however, in the previous study 77.8% of inhalant users were also using cannabis regularly. Therefore, comorbid cannabis use could have increased the deficits in WM integrity. Similar to our findings, unexpected increases in WM maturation have been seen with substance using adolescents in other studies. Bava et al. (2009) observed increased FA values among marijuana and alcohol using adolescents in the occipital lobe, internal capsule, and arcuate portion of the right superior longitudinal fasciculus. De Bellis et al. (2008) found higher FA values in the adolescents with alcohol use disorder in the corpus callosum. Similarly, Cardenas et al. (2013) also found that adolescent drinkers with no co-morbid substance abuse or externalizing disorder showed higher FA in WM tracts of the limbic system. There are several possible explanations for the observed higher AD values in adolescent inhalant abusers. First, it may be related to the presence of crossing fibers. Wheeler-Kingshott and Cercignani (2009) showed that a change in RD can cause a fictitious change in AD and vice versa in voxels characterized by crossing fibers. Second, higher AD values among the adolescent inhalant abusers may be due to compensatory repair processes. Previous animal and human studies reported that AD tends to be lower in the acute phase of brain injury whereas in the chronic phase, AD returns to normal or supranormal values, as the axon and myelin debris are gradually removed in the later stages which may be the mechanism behind the normalization of AD (Thomalla et al., 2005; Thomas et al., 2005; Budde et al., 2011). Third, increased AD in adolescent inhalant abusers may be related to the poor growth of neurofibrils, such as microtubules and neurofilaments, and the abnormal development of glial cells (Qiu et al., 2008). Finally, adolescent inhalant users may have more mature WM tracts relative to their conservative peers. Berns et al. (2009) found that engagement in dangerous behaviors was positively correlated with FA and negatively correlated with RD in right frontal WM tracts in the superior corona radiata and genu of the corpus callosum suggesting that adolescents who had higher impulsive traits may have more mature WM tracts. Therefore, increased WM maturation may indicate a risk for adolescent substance abuse rather than being a consequence of substance use. WM changes that we found in adolescent inhalant abusers compared to controls showed a more posterior distribution including the superior longitudinal fasciculus, inferior fronto-occipital fasciculus and inferior longitudinal fasciculus which also have projections to the prefrontal cortex. WM maturation processes follow a posterior to anterior direction (Lebel et al., 2008). Our results might have been influenced by this developmental asynchrony. When a person using inhalants becomes hypercapnic and hypoxic by rebreathing from a closed bag and unmyelinated axons seem to be more resistant to ischemia (Zammit et al., 2011), it might be advantageous for the anterior part of the WM tracts. Also, less myelination in the anterior part of the brain relative to the posterior part of the brain might lead to reduced neurotoxic effect of toluene in the anterior WM tracts.

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Adolescent inhalant abusers made more perseverative errors on the WCST than the controls. In other words, they had difficulties while switching between different rules or when the development of new rules was needed, observed by their significantly elevated number of perseverative errors. Adolescent inhalant abusers also showed deficiencies in attention and performance during the Stroop test, suggesting that adolescent inhalant abusers responded more impulsively. The Stroop task implicated the capacity to choose a weaker but task-relevant answer, regardless a stronger, but task-irrelevant one (MacLeod, 1991). Significant impairments on clinical neuropsychological (e.g., Stroop test, WCST) and experimental measures of executive control (e.g., Go/ No-go task, Eriksen flanker task, Simon task) have been identified in a range of dependent drug-using groups (Hester et al., 2010). In addition, the poor performance of the adolescent inhalant abusers on the Stroop test and WCST suggests that exposure to inhalants may lead to lower mental flexibility which could explain partially why executive deficits appear to negatively affect the treatment outcome (Turner et al., 2009). However, conclusions regarding the temporal relationship between inhalant use and executive functioning cannot be drawn from our data. Two possibilities suggested by this finding are that adolescents with less developed executive function tend to use inhalants more intensely and/or that inhalant use impairs executive functions. Prospective longitudinal studies are necessary to disentangle the causal relationships between inhalant use and cognitive performance. Posterior WM changes that we found in this study might represent part of the etiology of executive function performances in adolescent inhalant abusers. In line with this, Kennedy and Raz (2009) found that diffusion measures in posterior WM were associated with inhibition and task switching performance. These results are also consistent with the literature on top-down modulation of posterior brain regions during inhibition and attention tasks (Erickson et al., 2009). However, we did not found a significant correlation of AD values in the significant cluster with neuropsychological test performances. First, the lack of correlation may reflect the inadequate power in our study. In addition, the lack of direct correlation may be explained by the fact that several brain regions contribute to the features of neurocognitive performance and a disruption in one region may be compensated by other intact brain structures. Another possible explanation is that toluene may affect executive functions prior to detectable white matter abnormalities. For example, in rats, Duncan et al. (2012) showed that the deficit in rearing appeared to be progressive and being present by 4 weeks after initiation of toluene exposure while no changes in DTI parameters. Similarly, in humans a relatively short period of toluene exposure (8 months) results in impaired short-term memory and tactical performance (Ryu et al., 1998). This occurred although there were no apparent abnormalities on T1 or T2 weighted MR images, suggesting a disconnection between toluene induced behavioral deficits (including cognitive function) and WM pathology. The findings of this study should be interpreted considering the following limitations. An important limitation of this study is its crosssectional nature. Thus, it is unclear whether our findings resulted from inhalant use or pre-existed in individuals prone to inhalant use. Also there may be a third variable that causes a predisposition to myelin development dysfunction and inhalant abuse (e.g., the presence of belowaverage IQ or environmental factors). In addition, we recruited healthy control subjects who were matched with the abusers for age and sex. However, the adolescent inhalant abusers may have had other premorbid psychosocial factors that may have affected the results. For example, attention deficit hyperactivity disorder (ADHD) was reported to be associated with abnormalities in WM (Hutchinson et al., 2008) and an association between child abuse and reduction in the midbody of CC was also reported (Teicher et al., 2004). Both ADHD and child abuse are associated with problematic substance use. Thus, it is possible that predisposing factors might contribute to at least some of these observed differences. The frequent comorbidity of inhalant and cigarette use makes it difficult to examine the individual and combined

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effects of these substances on WM integrity. Longitudinal studies are needed to clarify these issues. Also, while the TBSS approach strives to avoid the problems of voxel based morphology related to partial volume effects, some of these issues may still remain. TBSS still has limitations in analyzing small fiber tracts, data with within-scan head motion, and regions of crossing fibers or tract junctions (Smith et al., 2006). Another limitation is that the measures of alcohol, past substance and inhalant use were self-reported, and under-report is a reasonable concern. A further limitation of our study is the lack of quantitative measures of lifetime smoking exposure. Therefore, we recommend an assessment of smoking status, e.g. using the Fagerstroem test for nicotine dependence (Heatherton et al., 1991) in future MRI studies. Finally, both the adolescent inhalant abuser and control groups were largely comprised of males, so our results cannot be generalized for females. In conclusion, we found that adolescent inhalant abusers had significantly higher AD values in left parietal, occipital and temporal WM than in the healthy controls and our sample of adolescent inhalant abusers showed worse performance in WCST and Stroop tests compared with controls. However, we only found discrete impairments in neuropsychological test performance between adolescent inhalant abusers and healthy controls and we were not able to show a direct correlation between WM alterations and neurocognitive performance. In the future, longitudinal studies are needed to evaluate brain abnormalities in adolescent inhalant abusers through methods that assess brain structure, function and connectivity. Role of funding source This research was funded by Ege University Science and Technology Application and Research Center (grant number 09/EGEBAM/001) which had no role in the design of the study, collection and analysis of data and decision to publish. Contributors Zeki Yuncu, Burge Basay, Omer Basay and Omer Kitis designed the study and wrote the protocol. Burge Basay, Omer Basay and Hozan Saatcioglu collected the MRI data. Nabi Zorlu, Pelin Kurtgoz Zorlu and Fazil Gelal undertook the MRI data analyses, and Nabi Zorlu and Zeki Yuncu wrote the first draft of the article. All authors have critically reviewed content. Conflict of interest The authors declared no conflict of interest. Transparency document The Transparency document associated with this article can be found, in the online version. References Allison W, Jerrom D. Glue sniffing: a pilot study of the cognitive effects of long-term use. Int J Ment Health Addict 1984;19:453–8. Ashtari M, Cervellione K, Cottone J, Ardekani BA, Sevy S, Kumra S. Diffusion abnormalities in adolescents and young adults with a history of heavy cannabis use. J Psychiatr Res 2009;43:189–204. Aydin K, Sencer S, Demir T, Ogel K, Tunaci A, Minareci O. Cranial MR findings in chronic toluene abuse by inhalation. Am J Neuroradiol 2002;23:1173–9. Bava S, Frank LR, McQueeny T, Schweinsburg BC, Schweinsburg AD, Tapert SF. Altered white matter microstructure in adolescent substance users. Psychiatry Res 2009; 173:228–37. Bava S, Jacobus J, Thayer RE, Tapert SF. Longitudinal changes in white matter integrity among adolescent substance users. Alcohol Clin Exp Res 2013;37:181–9. Berns GS, Moore S, Capra CM. Adolescent engagement in dangerous behaviors is associated with increased white matter maturity of frontal cortex. PLoS One 2009;4:e6773.

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