Am J Transl Res 2015;7(12):2795-2804 www.ajtr.org /ISSN:1943-8141/AJTR0015286 Original Article Neurochemical abnormalities in anterior cingulate cortex on betel quid dependence: a 2D 1H MRS investigation Tao Liu1,2*, Jianjun Li3*, Shixiong Huang2, Zhongyan Zhao2, Guoshuai Yang1, Mengjie Pan3, Changqing Li3, Feng Chen3, Suyue Pan1 1Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Depart- ments of 2Neurology, 3Radiology, People’s Hospital of Hainan Province, Haikou 570311, China. *Equal contribu- tors. Received August 30, 2015; Accepted December 12, 2015; Epub December 15, 2015; Published December 30, 2015 Abstract: The effects of betel quid dependence (BQD) on biochemical changes remain largely unknown. Individu- als with impaired cognitive control of behavior often reveal altered neurochemicals in Magnetic Resonance Spec- troscopy Imaging (MRSI) and those changes are usually earlier than structural alteration. Here, we examined BQD individuals (n = 33) and age-, sex-, and education-matched healthy control participants (n = 32) in an 2D 1H-MRS study to observe brain biochemical alterations in the anterior cingulated cortex (ACC) associated with the severity of BQD and duration of BQD. In the bilateral ACC, our study found NAA/Cr were lower in BQD individuals compared to the healthy controls, Cho/Cr and Glx/Cr were higher in individuals with BQD compared to the healthy group, but increase was noted for mI/Cr in BQD individuals only in the left ACC. NAA/Cr ratios of the right ACC negatively cor- related with BQDS and duration, NAA/Cr ratios of the left ACC negatively correlated with duration, Glx/Cr ratios of the right ACC positively correlated with BQDS. The findings of the study support previous analyses of a role for ACC area in the mediation of BQ addiction and mechanistically explain past observations of reduced ACC grey matter in BQD patients. These data jointly point to state related abnormalities of BQ effect and provide a novel strategy of therapeutic intervention designed to normalize Glu transmission and function during treating BQ addiction. Keywords: Betel quid, substance dependence, magnetic resonance spectroscopy, anterior cingulated cortex, N- acetylaspartate, myo-inositol, creatine, choline, glutamate Introduction The alkaloid arecoline contains parasympatho- mimetic properties which stimulate the musca- Areca nut (AN) can be chewed wrapping in a rinic and nicotinic receptors, therefore the AN betel leaf or together with tobacco (betel quid, primarily takes effects on the central and the BQ), and its composition differs among diverse autonomic nervous systems [3]. Frequent con- populations and areas [1]. In Hainan, China, BQ sumers indicate euphoria, which is a sense of is the combination of fresh areca nut and warmth, happiness, salivation, elevated alert- slaked lime such as aqueous calcium hydroxide ness, anti-migraine, palpitation and increased paste winded by betel leaves with no tobacco working capabilities [4]. BQ consumption is or any ingredients. BQ is broadly used by peo- related to a dependency syndrome, which con- ple of all ages around the globe, south-east sists of mild euphoria, enhanced concentration, Asia specifically. It ranked among the most postprandial satisfaction, relaxation and a wi- broadly consumed psychoactive substances thdrawal syndrome related to mood swings, worldwide following only nicotine, ethanol and insomnia, anxiety and irritability, and its severi- caffeine, and its consumer population accounts ty is similar to that of amphetamine [5]. The first for almost 10% of the world. The IARC review instrument measuring BQ dependence: the gave the conclusion that AN can be carcinogen- Betel Quid Dependence Scale (BQDS) was de- ic to people and may lead to cancers of the veloped and given initial validation by Lee re- pharynx, oral cavity, esophagus, the uterus as cently [6]. However, most studies regarding well as liver and biliary tracts [2]. Betel Quid chewing have been confined to epi- 2D 1H MRS investigation on betel quid dependence demiological and biological investigations so Materials and methods far [7, 8]. Researchers have conducted limited research to figure out the psychological and Ethics statement behavioral factors that cause people to start and/or maintain BQ consumption. To date, why The present study was approved by our re- people develop BQ chewing habit still remains search ethics review board of People’s hospital unknown. of Hainan Province, Haikou, China according to the Declaration of Helsinki. The consent form As a non-invasive imaging technique, Proton has been read and signed by each subject magnetic resonance spectroscopy (1H MRS) before being included in the study. measures specific compounds or metabolites within the tissue of interest. A number of bio- Inclusion and exclusion criteria chemical cerebral alterations had been dete- cted by drug abuse studies using 1H MRS. Native peoples that take BQs exclusively in Reduced N-acetylaspartate (NAA) and elevated Wanning City, Hainan province were included. myo-inositol (mI) were the most consistent Persons using Betel Quid with no tobacco more changes across drug class, while alterations in than 5 years were regarded as “individuals creatine, choline and amino acid transmitters using BQ without tobacco”. The criteria for BQD were also significant [9]. The research discu- group inclusion were as follows: (1) age: 18~60 ssed herein indicates that abused drugs may years; (2) persons with usage of BQ; (3) BQ affect an individual’s energy metabolism and chewer with BQD, the diagnosis was made ac- maintenance, neuronal health, inflammatory cording to BQD Scale (BQDS) by the BQDS > 4; processes, neurotransmission and cell mem- (4) persons without usage of different forms of brane turnover, and these alterations may un- tobacco at least for the last 1 years in order to derlie the neuropathology within cerebral tis- exclude the influence of nicotine; (5) not taking sue that leads to the behavioral and cognitive antidepressant drugs or psychotropic drugs; (6) impairments related to drug addiction subse- persons without self-claimed systemic illness- quently [9, 10]. However, rather than merely es (e.g. diabetes mellitus, cardiovascular dis- identifying the cerebral consequences caused ease, neurological disorder, thyroid, renal disor- by drug abuse, 1H MRS may track the progres- ders and epilepsy; (7) persons without present sion of disease and/or treatment. or recent history of any Axis I psychiatric and/or substance use diseases; (8) No abnormal man- The role of the anterior cingulate cortex (ACC) ifestation on conventional MRI; (9) left-hand- has led to its inclusion in many major theories ers; and (10) can read and write Chinese. of addiction, where it is believed to form part of The “control individuals” were persons without an inhibitory system that exercises control ov- using all forms of BQ, areca nut and tobacco. er reward-related behavior [11, 12]. As far as The exclusion criteria for control group were as we know, no magnetic resonance spectroscopy follows: (1) tobacco consumers; (2) persons (MRS) studies in individuals with BQD have using diverse forms of tobacco with no smoke been reported. However, based on findings in such as gutka and/or paan masala; (3) persons individuals with other drug abuse, we anticipat- with self-claimed systemic illnesses e.g. cardio- ed that patients with BQD would have similar vascular disease, neurological disorder, epilep- deficits related to the ACC to those which have sy, diabetes mellitus, renal disorders and thy- been observed in patients with alcohol abuse roid; (4) persons that have present or recent [13-15] or opiates abuse [16, 17]. The present history of any Axis I psychiatric and/or sub- study assessed the ACC in individuals with BQD stance use diseases; (5) currently using psy- using MRS aim to find the biochemical altera- chotropic drugs; (6) left-handers; and (7) can- tions, which underlie the neuropathology within not read and write Chinese. cerebral tissue that subsequently leads to dependency syndrome such as withdrawal, tol- Study participants erance, desire, concentration, and reduced ma- nagement of BQ addiction as well as persist- Finally, we included 33 BQD individuals and 32 ent chewing despite convincing indications of individuals as the control group selected from harmful influences. Wanning City of Hainan province, China. 2796 Am J Transl Res 2015;7(12):2795-2804 2D 1H MRS investigation on betel quid dependence Figure 1. Placing region of interesting in right ACC and measuring the metabolite ratio. Questionnaire MRS data processing and analysis We distributed questionnaires regarding infor- MRS data were post processed with Syngo mation of age and sex, educational status, spectroscopy software (Siemens Healthcare, monthly income, daily dosage of BQ, duration of Erlangen, Germany). The postprocessing BQ chewing habit and duration time of quid included the following steps: frequency shift placement in mouth in simple Chinese to all correction by using the residual water signal, participants (n = 65). Since wine plays a signifi- removal of residual water signal, filtering the cant part in Chinese society, alcohol use in the data by a Hanning low-pass filter with a width of past 30 days of the participants were also 400 ms, zero-filling the data to 2048 points required, as well as the number of drinks per from 1024 points, Fourier transformation, auto- occasion and average frequency of drinking. matic baseline correction, automatic phase correction (manual adjustment were done if MRI data acquisition automatic correction did not work well for some cases) and curve fitting of the signal peak at Structural MR and 1H-MRS were performed around 2.0 ppm as NAA, 2.4 ppm as Glx, 3.0 using a Siemens Verio3T MRI scanner and a 6 ppm as Cr, 3.2 ppm as Choline, and 3.60 ppm channel Sense head coil. We conducted a rou- as Myo-inositol respectively using Gaussian tine structure MR scan to rule out gross cere- line-shape. bral pathology. Anatomical images were Statistical analysis acquired with a high-resolution T1-weighted MPRAGE sequence for the acquisition of 3D All data were compared between the two volume images.