The effects of early girl-child marriage in Mutasa District- Manicaland Province: A cases of Samanga 'A' Ward in Honde Valley

Offard Kanjanda and Getrude Vongai Chiparange

European Scientific Journal. 12.11 (Apr. 15, 2016): p539. From InfoTrac Science Collection 2017.

Abstract

Girl-child marriages in the developing countries of the world have caused a lot of suffering on the girl-children. According to Giddens (2009) issues of poverty, cultural practices, and political instability and gender inequalities have been noted as some of the major causes of girl-child marriages in developing countries. The situation has caused great concern to the communities of the global village because of the serious damages on the victims. Gage (2011) states that the status quo was exacerbated by the historical gender inequalities that continue to exist within families systems and place the girl-child an inferior citizen in her country. The girl-child remained vulnerable and segregated particularly, in educational advancement which is the key to self-empowerment, knowledge and skills development because of the high rate of girl-child marriage in the area. It appears there is little significant research on the matter in the developing countries. In order to establish some strategies to promote the girl-child's life-skills a study was conducted in Samanga 'A' in Honde Valley in Manicaland Province-Zimbabwe. From a population of 1500 a sample size of 100 participants was selected using the cluster, systematic and purposive techniques because of their appropriateness to the two paradigms (Punch, 2009). Cluster technique was employed because the participants lived in different kraal-heads. The systematic technique was engaged because the participants were selected from every tenth household in each of the five kraal-heads. The purposive technique was used to select the influential participants such as local community leaders and the victims of early girl-child marriage. Marshall and Roseman (2006) support that purposive technique in qualitative study because the participants are powerful sources of information that is needed. Both qualitative and the quantitative paradigms were used in the study because some aspects of the study required simple response, while some of the questions demanded the participants' in-depth knowledge on the problem (Newman, 2010). A case study methodology was adopted because of its ability to focus on a specific issue in a concerned area (Croll, 2010). Data were collected through the use of direct observation, questionnaire with both open and closed ended questions and interviews. The data were descriptively analysed. The research revealed that the prevalence of early girl-child marriage was perpetuated by the society's cultural deprivation ideology which has since seen the girl-child discriminated in her efforts for self - empowerment through education. The study recommended that there is need for the government to enforce and implement gender-sensitive policies which aim at protecting the girl-child in-order to enhance economic, social, cultural and political transformation for sustainable development in the community.

Keywords: Girl-child marriage, discriminate, self-empowerment, transformation, sustainable, gender-sensitive perpetuate and ideology

Background to the Study

According to the United Nations Children's Education Fund (UNICEF) (2005), globally 36% of women aged between 20 and 24 were married or in union before they reached 18 years of age. United Nations Educational, Scientific and Cultural Organisation (UNESCO) (2004) also states that globally, 39 million girls aged 14-15 in developing countries do not reach secondary education due to several reasons that include early marriage. Sweetman (2006) also states that up to half of the girls in developing countries are mothers before they turn 18 years. Zimbabwe, as one of the developing countries is experiencing similar cases of early girl-child marriage.

There is much concern over the involvement of girls who are married before the national maturity age of 18 years since they still have dreams of acquiring educational achievements and finding employment to end the cycle of poverty in their families. The affected children experience a lot of problems ranging from social, psychological, educational, emotional and even physical. Many of them usually die before they reach the age of 45 years (Sweetman, 2006). Surveys carried out in some African countries offer alarming examples of early girl-child marriage. The table below shows the data about girls who are married before reaching 18 years in Africa.

According to Gage (2011) and Kanyi, (2008) the causes of early girl-child marriages in these countries are numerous and unsounded. These include poverty and cultural factors. The Central Intelligence Agency (2011) also argues that most of these countries are bordered by other countries that have history of wars and conflicts. Parents in such countries marry off their girls in order to protect them from rape and abductions from rebels.

According to the Zimbabwe Demographic and Health Survey (ZDHS) of 2010, the country's current economic and political situations have forced many families to violate children's rights, especially those of the girl-child. The table below shows the girl-child marriage prevalence in Zimbabwe:

From the statistics above there is evidence that girl-child marriage is prevalent in Zimbabwe. According to ZDHS' (2010-11) research findings, revealed that about one in four teenagers aged between 13 and 19 have already begun child bearing because of poverty, religious belief systems and cultural practices. The BIAAG report (2012) also confirmed that at one school in Midlands, ten girls in Forms 2 and 3 left school due to early marriage while in Mashonaland East, 13 girls dropped out of school for the same reason in that year. From these few examples it is clear that there are many more cases of such nature which are just swept under the carpet in order to protect some few individuals to the expense of the innocent girl-child whose life is put to waste forever.

The Standard of 21 October, 2012 also reported on increasing cases of girl-child marriages in Mutasa District in Manicaland Province where nearly 50 girls drop out of school each year and get married to older men. This was revealed by the Education Officer, Mr. Derek Duma during the commemoration of the International Day of the Girl Child. Cultural beliefs were cited as one of the causes of the girl-child marriage in the area. Speaking at the same occasion, Headman Mandeya of Honde Valley said that some girls were married off to older men to appease avenging spirits. In Samanga 'A' Ward in Honde Valley, the issue of early girl-child marriage has reached an alarming level and the effects are just serious such that there is need to intervene in order to rescue the girl-child from further abuse as a result of early marriage. According to Mbiti (2008) child neglect has led to psychological, social, emotional, physical and educational problems. Current interventions lack commitment and coordinated action to eradicate this practice. Thus, this study sought to establish strategies that can help to eradicate the girl-child marriage in Mutasa District, with special emphasis to Samanga "A" Ward of Honde valley in Manicaland-Zimbabwe.

Statement of the problem

The status quo in Samanga Ward "A" is very influential the early girl-child marriage due to their cultural and religious beliefs. This was intensified by the historical inequalities that exist between men and women about the position the position of girls in this community society. This practice has caused untold girl-child suffering which this study will reveal.

Purpose of Study

The purpose of the study was to analyse the causes and effects of early girl-child marriage so as to eliminate all forms social inequalities that deprive the girl-child of her right to a decent life in the society.

Objectives of the Study

The study was guided by the following objectives:

1. To identify factors leading to increased early girl-child marriages in Samanga "A" Ward of Honde Valley, in Manicaland.

2. To establish the effects of early girl-child marriage in Samanga A ward.

3. To suggest possible strategies to reduce early girl-child marriage in Samanga 'A' Ward.

Research Questions

1. Why does early girl-child marriage exist in Samanga 'A' Ward in Honde Valley?

2. What are the (a) educational, (b) the social (c) psychological and (d) physical effects of early girl-child marriage on the girl-child?

3. What strategies can be employed to mitigate early girl-child marriage in Samanga A Ward?

Theoretical Frameworks

Although in qualitative approach the theory is grounded, it was important for the researchers to take a position on which theory or theories could guide the research. In this research the researchers used the systems and the grounded theories.

Systems theory

Barret (2010) believes that a system is a unified whole with interrelated parts. In this research, the researchers used the theory to find out how the girl-child lived with the new systems when they were married. This became the main purpose of this research in which the researchers would want to unveil the challenges faced by the girl-child as a result of early marriages. In the African culture, systems are important to promote collectivism and to maintain the value of "Ubuntu" within individuals (Saidel, 2010). It is within the context of this value that we would want to find out the extent to which this concept is inculcated in the community so that they are not divorced from their families and cultures.

Grounded Theory

It asserts that the researcher gets into a research without a concrete theory for the problem. As the researcher interacts with the participants and gets new knowledge, insights and concepts on the problem through continuous generation of new data, he/she establishes some theories from which to choose the most appropriate one(s) for the research (Punch, 2009). In this study the researchers used the grounded theory through immersion and debriefing with the participants as well as triangulation of instruments.

Research methodology

Qualitative/case study method

The qualitative paradigm was employed in this study because it enabled the researchers to collect in-depth information on what the community say or do in their natural settings, (Croll, 2010 Seidel, 2010). The design enabled the researchers to focus on the affective, cognitive and behaviour domains of the participants on the effects of early girl-child marriage. The question of perceptions and attitudes is an abstract concept and requires direct inquiry so as to unravel perceptions because they lie at the heart of the participants (Gage, 2011; Punch 2009). The researchers employed a qualitative/ case study because it was an appropriate method of studying of studying phenomena using a thorough analysis of an individual case among many. Marshall and Roseman (2006) contend that a case study provides a unitary character to the data being studied through triangulating facts that are derived from a variety of instruments. Therefore, the richness of a case study lies in its ability to provide in-depth understanding of important aspects of a new problematic area. Although this approach was successfully used the researchers experienced some difficulties because of multiplicity of perceptions resulting from the multiplicity of cultures in this area. To overcome this short coming, the researchers borrowed some quantitative techniques for data presentation and analysis.

Population, Sample and Sampling Procedures

During the time of study, the population of Samanga "A" Ward was 1500 families, statistical numbers provided by the Ward Councillor and kraal-heads. The sample constituted 100 participants (60 females and 40 males) for the research. The females were more than males because they were always available when the study was carried and most women had the responsibility of working in the banana and coffee plants to support the family. Because the researchers worked with different villages, stratified and systematic sampling techniques were used to make the sample. Using the systematic sampling technique, every 100th house was selected. Purposive sampling was used to select six kraal-heads because of their positions and influence in the community.

Research Instruments

In order to collect data the researchers used the questionnaire, direct observation and interviews.

Questionnaire

The questionnaire had closed and open-ended questions to solicit in-depth understanding of the phenomena (Meggitt, & Grenier (2011). Questionnaires were easy to complete because they were self-administered and participants were given ample time of seven (5) days to complete the questionnaires. Among its many advantages, for the purpose of this study the questionnaire allowed the participants time space to express their views to the questions.

Direct Observation

The researchers used direct observation to generate data in the participants' natural environment. This was rich because it enabled the researchers to record what was happening in the real world of the participants (Johnson, 2007). The researchers also observed the young women of different age groups struggling to do some manual work, daily activities and health outlook. This facilitated for accurate and unbiased data.

Interviews

For this particular study, the interview was the most prominent data generation tool that allowed the participants to describe the phenomenon under study (Punch, 2009). Interviews were regarded very reliable way of accessing people's perceptions, meanings, definitions of situations and their constructions of reality (Marshall & Rossman, 2006). In order to understand others the researchers needed participants' constructions of reality to ask them -and ask participants in ways that they can provide in-depth description of the phenomenon.

Data Collection Procedures

In order to access entry into the field of study the researchers sought permission from the Headman, the kraal-heads, Ward Councillor and Ward chairperson. The councillor provided the researchers with statistical data for the population in the area. The data were collected using questionnaire, direct observation and semi-structured interviews.

Data Presentation and Discussion

Qualitative data were carefully coded and presented descriptively while quantitative data were presented in tables and supportive literature was added.

Data generated from the participants through interviews and questionnaire to define early girl-child marriage, the research findings indicated that they had a common understanding of the definition. However, they came up with three major definitions as illustrated on the above table.

The responses above show that the community was aware of what early girl-child marriage was. Croll (2010) and Giddens (2009) shared the same sentiments that... what society believes in is what it adopts as its culture, and this belief is passed on to the next generation. This suggests that early girl-child marriage is a culturally accepted socialization process in certain societies, despite it being a violation of human rights. However, whether with or without the consent of the child, girl-child marriage remains a serious violation of the child's rights because the young girl is immature to make decisions (Sweetman, 2006; & Croll, 2010).

Item 2: Is early girl-child marriage a human rights problem?

Data generated from the participants to give perceptions on whether early girl-child marriage is a human rights problem reflected that the community in this area is aware that girl -child is a human being whose rights need to be observed and respected. With the Zimbabwe government making efforts to educate and empower its community on the rights of the girl-child, the research findings revealed that there still remain some gaps as evidenced by the attitude and practices by some members of the community who participated in this study. To confirm this finding one of the interviewed men stated that:

Headman 2

Hapana chikonzero chokusiya vakadzi vachingo mbeyambeya vasinana

kuroorwa nokuti tinoda kuvedzera uwandu hwevanhu munharaunda yedu.

There was no reason to leave females to loiter around when they

unmarried because we want to increase the population in our area and

labour in the families.

Among the interviewed participants one of them came up with a strategy to make people aware of the existence of the girl-child in the community. He stated that:

Councillor: There is great need for rescuing the innocent souls from

further abuse so that they also enjoy their lives as the first class

citizens in the planet. It is therefore important that Gender-based

Movements step their awareness campaigns in order to give more

education and information to the communities of Zimbabwe.

From the citations above it reflects that lack of awareness and knowledge on human and children's rights within communities will cause more damage to the girl-child. This is congruent with the findings of the Universal Declaration of Human Rights (1948) which is calling for the abolition of bride price, elimination of child marriage and betrothal of young girls before the age of puberty.

Item 3: Society's attitudes towards early girl-child marriage

The research findings clearly indicated that in Samanga ' A' Ward people had mixed attitudes towards the early girl-child marriage. People have mixed attitudes on the early girl-child marriage. From the data generated from the participants the following comments depicted the community's attitudes towards early girl-child marriage: It is morally acceptable; We are comfortable with the practice; It is the girl's choice to get married; It is a bad practice on human beings; There is great need for positive change; No-one cares about it; We sympathise with the girl-child.

The greater part of the responses condemned the early girl-child marriage as a shameful practice by some individuals who would want to justify protection of their own interests to the expense of the innocent girl-child. The implication here is that most communities do not condone such practice because it is a violation of human rights and is unjust.

Interviews carried with the parents and teachers on the educational effects of the early girl-child marriage showed that some children are not allowed to attend school because the parents fear that the daughters will be taken by some other males. Additionally, one of the teachers stated that:

Teacher 3

"In my class some girls perform very poorly at school and others are not provided with adequate educational resources such as text books and exercise books. This practice practice increases illiteracy levels in the child."

The citations above pointed out that the girl-child is neglected from receiving the desired academic achievement. There seems to be a strong belief that a certain gender (the girl-child) should not reach a certain academic level so that she remains a rich source for man's abuse. The cultural deprivation model assumes that this pattern of thinking is very dangerous because the community may find it difficult and irrelevant to accept change when, in fact, this is its culture (Haralambos and Holborn, 2011). This increases the girl-child's chances of remaining a second class individual and dependent in the society (Oxfam, 2004 & UNICEF, 2000). From the Interpretivism point of view, some societies seem to have developed some neurotic behaviour simply because they were socialized in such culture(s). From the functionalism perspective, for a community to function effectively and productively for sustainable economic and social development, every member of the society should be an effective participant; and with resources as the social capital, shared equally among members (Giddens, 2009. Therefore, awareness campaigns against such behaviour should be scaled up so that the girl-child also benefits from educational resources so that she is able to determine her own future without disturbances and to live as a first class citizen in her own country.

Item 5: The social effects of the girl-child marriage

In order to show the social effects of the girl-child data generated from the participants depicted serious social effects on the girl-child as she gets married prematurely. There is evidence from direct observation that the girl-child's pattern of life in the marriage relationship was characterized by poverty, sorrow and anguish and therefore a slave in a marriage relationship.

Item 6: The physical effects of girl-child-marriage

Of all the twenty girls who were interviewed research findings showed that their responses were common. However, one of the girls who was physically abused stated that:

Girl-child 12

I was married at the age of 12 when I was doing Grade seven. My husband

was older than my father. He asks me to fetch firewood from a far

mountain with this pregnancy and I do all the house-hold chores for the

whole family. 'Ndinorara ndaneta chaizvo,' I go to sleep tired.

Similarly, one other girl sorrowfully stated that:

Girl 16:

I blame my father who pushed me into this mess. I am the fourth wife married to a Johanne Marange man who is almost 60 years and I am only 13 years. Most of the duties that are carried by a wife are very difficult for me to do. The family spends most of their time in the banana fields weeding and fetching firewood. This pains me because the work load is too heavy for my age.

From these citations there is a reflection that most of the girl-children are not enjoying their marriage relationships. From the data generated some of them confessed that they shall eventually go back home and continue with their educational studies.

The findings of this research were in agreement with Haralambos and Holborn (2011) who stated that the physical effects of girl child marriage negatively impact their lives since these marriages are not done to the best interest of the child. The CRC, Article 24 provides a basis for evaluating the laws and practices of States with respect to the protection of children. To pursue the best interest of children, parents and governments are responsible for protecting their children's health, education, development and overall well-being to the best of their capacities. Since early girl-child marriage disturbs the girl-child's health, particularly her sexual and reproductive health, which often results in maternal mortality and morbidity due to early pregnancies, the governments should take effective and appropriate measures to abolish such practice that prejudice the health of the girl-child.

Conclusion

The following conclusions were drawn from this current study:

* Early girl-child marriage is prevalent in societies whose cultural beliefs and attitudes are difficult to change. Early girl-child marriage is a culturally gender practice which discriminates equal opportunities and places the girl--child a second class citizen in her society. The research study revealed that early girl-child marriage as a cultural gender practice is prevalent in Samanga 'A' Ward.

* The prevalence of early girl-child marriage is greatly perpetuated by the society's mixed attitudes which have since seen the girl-child discriminated in her efforts for self-empowerment and self -emancipation through education. Education as a strong tool to acquire knowledge and life skills is regarded as the boy child's right to acquire.

* Dominance of patriarchal attitudes coupled with autocracy within some family systems make the girl child an inferior subsystem.

* Early girl-child marriage is a deliberate attempt by some individuals who intend to protect and achieve their own interest as evidenced in the Johane Marange sect.

* Some men take advantage of the girl child's poverty to cheat her into marriage by promising that she will be provided with everything.

* Early girl-child marriage is a violation of children's rights because the child is deprived of her personal freedom, effective social interaction and personal achievement for sustainable social, economic, spiritual and psychological development

* he effects of early girl-child marriage are generally a result of poor family backgrounds. When parents fail to educate the girl child no one else will educate her and it means her future life is doomed

Recommendations

The research recommended that:

* There is great need to make it mandatory that every girl-child must attend and attain secondary education which will make her concentrate on school work, socialise on the effects of early marriage and gains life skills for economic growth and sustainable development.

* Gender inequality should be a teaching and examinable subject from primary to secondary education so that the society is educated for continued sustainable economic, moral, social, political and spiritual transformation and development.

* More community awareness and empowerment programs should be intensified in order to reach all corners of the country and follow up by the government should be done to check and evaluate on the effective implementation of the programs. This effort will change undesirable behaviours and attitudes on the girl-child.

* Communities, traditional leaders and religious groups should be capacitated with knowledge and skills that enable them to enforce mechanisms that eliminate the practice of girl-child marriage.

* Recovery and social reintegration services should be available for all girl child victims of girl child marriage to enable them to regain their damaged ego for continued sustenance of life.

* Reporting mechanisms available to children and their representatives must be safely accessible to enable the reporting of girl child marriages, taking into account the special difficulties for the child and their advocates caused by the dominant approval of the practice. Reporting must lead to appropriate action to protect children in their best interest.

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Mr. Offard Kanjanda

Ms. Getrude Vongai Chiparange

Lecturer, Zimbabwe Open University

Table 1: Statistics of Girl Child marriages in Africa

Country       Statistics of the country

Ethiopia                   49%

Mali                       65%

Mozambique                 57%

South Africa               38%

Togo                       39%

Uganda                     54%

Zimbabwe                   33%

Source: Demographic Health Survey (DHS) data from 1995 to 2010

Table 1.2 Statistics of girl child marriage in Zimbabwe by province

Province             Prevalence of girl-child marriage

Mashonaland Central                  50%

Mashonaland East                     36%

Mashonaland West                     42%

Masvingo                             39%

Matabeleland North                   27%

Matabeleland South                   18%

Bulawayo                             10%

Harare                               19%

Manicaland                           30%

Midlands                             31%

Source: UNFPA report 2010

Item1: Which one of these definitions best fits early girl -child

marriage?

Variable                                     Number of Respondents

                                             Male   %   Female   %

Early girl-child marriage is when a girl is  35     35   35      35

forced to marry at the age that is below 18

years.

Early girl-child marriage is when a girl is   5      5   23      23

married without her consent.

Early girl-child marriage is when a young     0      0    2       2

single mother marries again

TOTAL                                        40     38   60      62

Variable                                     Total  %

Early girl-child marriage is when a girl is   70     70

forced to marry at the age that is below 18

years.

Early girl-child marriage is when a girl is   28     28

married without her consent.

Early girl-child marriage is when a young      2      2

single mother marries again

TOTAL                                        100    100

Item 4: Educational effects of early girl-child marriage.

THE EDUCATIONAL EFFECTS                            FREQUENCY

Not allowed to attend school for fear

of being proposed by other                            84%

males

Poor academic performance.                            78%

Not provided with adequate educational resources.     79%

Increase of illiteracy in the child.                  56%

Alcohol induced mitochondrial oxidative stress and alveolar macrophage dysfunction

Yan Liang, Frank L. Harris and Lou Ann S. Brown

BioMed Research International. (Annual 2014):

DOI: http://dx.doi.org/10.1155/2014/371593

Copyright: COPYRIGHT 2014 Hindawi Publishing Corp.

http://www.hindawi.com/journals/bmri/

Abstract:

An alcohol use disorder increases the risk of invasive and antimicrobial resistant community-acquired pneumonia and tuberculosis. Since the alveolar macrophage (AM) orchestrates the immune response in the alveolar space, understanding the underlying mechanisms by which alcohol suppresses AM phagocytosis is critical to improving clinical outcomes. In the alveolar space, chronic alcohol ingestion causes severe oxidative stress and depletes antioxidants which are critical for AM function. The mitochondrion is important in maintaining cellular redox balance and providing the ATP critical for phagocytosis. The focus of this study was to understand how alcohol triggers mitochondrial reactive oxygen species (ROS), stimulates cellular oxidative stress, and induces AM dysfunction. The current study also investigated the capacity of the mitochondrial targeted antioxidant, mitoTEMPOL (mitoT), in modulating mitochondrial oxidative stress, and AM dysfunction. Using in vitro ethanol exposure and AMs from ethanol-fed mice, ethanol promoted mitochondrial dysfunction including increased mitochondrial ROS, decreased mitochondrial membrane potential, and decreased ATP. Treatment with mitoT reversed these effects. Ethanol-induced decreases in phagocytosis and cell viability were also attenuated with mitoT. Therefore, antioxidants targeted to the mitochondria have the potential to ameliorate ethanol-induced mitochondrial oxidative stress and subsequent decreases in AM phagocytosis and cell viability.

1. Introduction

Both acute and chronic alcohol consumption have well-documented effects on the immune system leading to increased susceptibility to community acquired pneumonia and tuberculosis [1]. When subjects with an alcohol use disorder get pneumonia, they are more likely to be infected with serious Gram-negative bacteria [2] and these increased risks occur even in those who do not meet the diagnostic criteria for an alcohol use disorder [3]. This results in a higher rate of intensive care use, longer inpatient stays, higher healthcare costs, and a 2-4 times greater mortality rate [4]. There is also an increased risk of ventilator-associated pneumonia which worsens the morbidity and mortality rates [5]. Alcohol abuse is also associated with a 2-3-fold increased risk of the acute respiratory syndrome (ARDS), representing ~50% of all ARDS cases with an average age of 30-35 3]. For subjects without a history of alcohol abuse, pneumonia will lead to sepsis in ~35% of the cases and ~30% will progress to ARDS. In contrast, pneumonia will lead to sepsis in ~60% of the cases if the subject has a history of alcohol abuse and 70% will progress to ARDS [3].

A seminal feature is that chronic alcohol abuse causes severe oxidative stress in the fluid lining the alveolar space, which includes the depletion of the critical antioxidant glutathione (GSH) and oxidation of the GSH/GSSG redox state by ~40 mV in subjects with an alcohol use disorder [6,7]. GSH depletion and oxidation within the alveolar space are particularly critical for alveolar macrophages (AM) since they are constantly bathed by this fluid and depend on this GSH pool for cellular uptake and protection against the oxidative stress generated during immune responses. Residing at the inner epithelial surfaces of airway and alveoli, AMs are the only lung phagocytes exposed directly to the environment. Therefore, AMs represent the first line of cellular defense in the lower respiratory tract [8]. However, oxidative stress can impair AM phagocytosis [9, 10]. In addition to impaired clearance of microbes, impaired phagocytosis can cause insufficient clearance of dying or dead cells and lead to pathological inflammation. Therefore, alcohol-induced oxidative stress can be a critical contributor to pulmonary pathophysiology, risk of infection, and contribute to the increased risk of tissue injury associated with ARDS.

There are multiple cellular sources of reactive oxygen species (ROS) including the mitochondria, the cytochrome P450 family, xanthine oxidoreductase, peroxisomes, cyclooxygenases, lipoxygenases, and the family of NADPH oxidases [11]. The consequences of the ROS depend on the type of the ROS generated, the amount of ROS, and where it is generated. Under resting conditions, the majority of the cellular ROS generated is derived from the mitochondria where ~90% of the oxygen used by a cell is consumed during energy metabolism [12]. In this mitochondrial process, nicotinamide adenine dinucleotide (NADH) is oxidized to support electrochemical coupling of oxidative phosphorylation and ATP synthesis [13-16]. However, respiration also generates ROS such as superoxide anions ([O.sub.2.sup.*-]), hydrogen peroxide ([H.sub.2][O.sub.2]), and hydroxyl radicals ([sup.*]OH). To protect against the ROS generated during respiration, mitochondria also maintain redox balance through numerous ROS defense systems including mitochondrial manganese superoxide dismutase (MnSOD), GSH, thioredoxin 2 (Trx2), and catalase [17]. Neutralization of mitochondrial ROS is critical for mitochondrial function and, ultimately, cellular functions but low-level concentrations of ROS are also required for signal transduction [18]. During respiration, the NADH is oxidized to [NAD.sup.+] and the [NAD.sup.+]/NADH ratio has been recognized as a key regulator in energy metabolism, aging, and immunological functions [19]. For example, decreases in [NAD.sup.+] or in the [NAD.sup.+]/NADH are associated with increased production of superoxide by the mitochondria and subsequent alteration of the mitochondrial redox system [20-22].

Alcohol metabolism can interrupt this complex integrated redox system within the mitochondria. Whether it is metabolized by alcohol dehydrogenase or cytochrome P450, the primary metabolite produced during alcohol metabolism is acetaldehyde. Within the mitochondria, acetaldehyde is metabolized by mitochondrial aldehyde dehydrogenase (ALDH2) [23] which uses [NAD.sup.+] as a cofactor. Thus, acetaldehyde metabolism decreases mitochondrial [NAD.sup.+] pools and increases NADH. The resulting decreases in the [NAD.sup.+]/NADH ratio and subsequent increases in mitochondrial ROS can change the mitochondrial redox balance leading to cellular oxidative stress and damage. Our research team has previously demonstrated that chronic alcohol ingestion resulted in impaired phagocytosis by AMs and chronic oxidative stress was central to the impaired immune functions [9, 10], while alcohol-induced upregulation of ROS through NADPH oxidases is linked to impaired phagocytosis; we speculated that there was also a role for alcohol-induced mitochondrial ROS generation in impaired AM phagocytosis. The results presented in this paper demonstrate that chronic alcohol ingestion in a mouse model induced mitochondrial ROS generation and mitochondrial dysfunction which contribute to impaired AM phagocytosis. Treatment with mitochondrial specific antioxidants reversed mitochondrial dysfunction and restored phagocytosis.

2. Materials and Methods

2.1. Mouse Model of Chronic Ethanol Ingestion.

All animal studies were performed in accordance with the National Institutes of Health guideline outlined in the Guide for the Care and Use of Laboratory Animals. All described protocols were reviewed and approved by the Emory University Institutional Animal Care and Use Committee. Mice (C57BL/6; age 6-8 weeks) were fed standard laboratory chow ad libitum with incremental increases of ethanol in the drinking water over 3 weeks (5%/week) to a final concentration of 20%. Mice were maintained at 20% ethanol (EtOH) in the drinking water for 10-12 weeks (n = 5/group) [24, 25]. The controls were pair-fed in order to control for the calories due to EtOH as well as any differences in food intake. The weight of the chow consumed by the mice with ethanol in the drinking water is routinely determined and this historical data was then used to establish a pair-feeding model for the controls. This regimen produced clinically relevant elevations in blood alcohol concentrations of 0.12% [+ or -] 0.03, as published by our group [26] and others [27, 28]. After euthanasia, tracheas were cannulated and a bronchoalveolar lavage (BAL; three 1 mL of saline) performed. Mouse AMs (mAMs) were then isolated from the fluid by centrifugation at 1000 xg for 10 min. After differential staining with Diff-Quik (Dade Behring, Newark, DE) and counting with a hemocytometer, the cell population was determined to be ~95% alveolar macrophages. The cell pellet was resuspended in RPMI 1640 medium containing 2% FBS and 1% penicillin/streptomycin and cells were incubated at 37 [degrees]C in 5% C[O.sub.2] atmosphere before the experiments outlined below were performed.

2.2. MH-S Cell Culture and EtOH Exposure.

The mouse AM cell line, MH-S (American Type Culture Collection, Manassas, VA), was used as a model system for studying the direct effects of EtOH exposure in vitro. Cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin and incubated at 37[degrees]C in a 5% C[O.sub.2] atmosphere. MH-S cells were treated with 0.2% EtOH for 5 consecutive days with the media changed daily. This EtOH concentration (0.2%) is representative of the blood alcohol content (BAC) when a 120 lb person consumes 5 drinks at a single sitting [28-30]. During the last 24hr of the 5d EtOH treatment, some cells were also treated with the mitochondria-targeted antioxidant mitoTEMPOL (mitoT, 100 [micro]M) [31].

2.3. Measurement of Intracellular ROS Generation.

After EtOH exposure, the cellular ROS sensitive probe CM-[H.sub.2]DCFDA (Invitrogen; Carlsbad, CA) and the mitochondrial superoxide probe mitoSOX (Invitrogen; Carlsbad, CA) were added to the medium (10 [micro]M, 30 min, 37 [degrees]C). Cells were then harvested, washed, and resuspended in phosphate buffered saline (PBS) for FACS analysis by BD Canto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). CM-[H.sub.2]DCFDA and MitoSOX were excited at 488 nm and detected at 530 [+ or -] 15 nm or 585 [+ or -] 42 nm, respectively. Data analysis was performed using Flowjo (http://www.flowjo.com/).

2.4. Measurement of Mitochondrial Membrane Potential.

After the different treatments, the mitochondrial membrane potential was determined by incubating the cells with tetra-methylrhodamine, ethyl ester (10 nM, 30 min, 37 degrees]C; TMRE; Sigma, St. Louis, MO). This cell-permeable, positively charged, red-orange fluorescent dye is readily sequestered by active mitochondria due to the relative negative charge of the fluorophore. However, depolarization of the mitochondrial membrane results in a failure to sequester TMRE. Cells were then harvested, washed, resuspended in PBS, and analyzed by FACS analysis. Data analysis was performed using Flowjo.

2.5. Measurement of Mitochondrial ATP Production.

ATP production was measured by a plate reader bioluminescence assay following the manufacturer's instructions (abcam, Boston, MA). In brief, MH-S cells were harvested after the appropriate exposures, stained with Diff-Quik (Dade Behring; Newark, DE), and counted using a hemocytometer. 10 [micro]L of a cell resuspension ([10.sup.3] - [10.sup.4] cells) was mixed with 100 [micro]L of the reaction mix for 5-10 min and then read in a luminometer. The ATP values were normalized to the cell count for each sample.

2.6. Colorimetric Assay for Measuring the Mitochondrial Ratio of NAD/NADH.

EnzyChrom [NAD.sup.+]/NADH assay kit (Bioassay Systems; Hayward, CA) was used to determine the mitochondrial ratio of [NAD.sup.+]/NADH. In brief, mitochondria were isolated from MH-S cells or mAMs using a Mitochondria Isolation Kit (Thermo Fisher Scientific, Rockford, IL). [NAD.sup.+] and NADH were then extracted with the extraction buffer provided in the assay kit, mixed with assay buffer, and absorbance-read at 565 nm.

2.7. Measurement of Phagocytosis and Cell Viability.

To determine the phagocytic capacity of macrophages, pHrodo Red S. aureus bioparticles conjugate (Invitrogen, Carlsbad, CA) was added to the culture media according to the manufacturer's recommendations with ~2 x [10.sup.6] cells per 2 mg vial of pHrodo-labeled bioparticles. Cells were incubated with the pHrodo labeled bioparticles for 2 hrs and then collected for FACS analysis and data analysis by Flowjo. This phagocytosis assay is based on the fact that there is a minimal fluorescence signal when the pHrodo Red S. aureus bioparticle conjugate is adherent to the outer surface of the phagocyte. Once the S. aureus is internalized and incorporated into the acidic environment of the phagosome, the bioparticle conjugates emit a strong red fluorescence. Internalization was verified by live cell confocal imaging (Olympus FluoView FV1000, Center Valley, PA). To assess changes in viability due to ethanol, MH-S cells were stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and Propidium Iodide (PI) (Invitrogen, Carlsbad, CA) before analysis by flow cytometry.

2.8. Fluorescence Microscopy and Image Analysis.

mAMs isolated from EtOH-fed and control mice were cultured overnight in 8-well cover glass bottom chambers (Lab-Tek; Scotts Valley, CA) with RPMI 1640 medium containing 2% FBS and 1% penicillin/streptomycin. Some mAMs were also treated with 500 [micro]M mitoT for 24hrs. ROS probes CM-[H.sub.2]DCFDA (10 [micro]M) or mitoSOX (10 [micro]M) was added to the media and images were taken after a 30 min incubation. Images were acquired with Olympus FluoView FV1000 Confocal Microscope using a 63 x oil objective. Images were viewed and analyzed by FV10-ASW 2.0 (Olympus, Center Valley, PA). For mitochondrial morphology analysis, acquired images were subjected to particle analysis using ImageJ Particle Analyzer (National Institutes of Health (http://rsbweb.nih.gov/ij/)). After thresholding, individual particles (mitochondria) were analyzed for area, perimeter, circularity (4[pi] x Area/([perimeter.sup.2])), and the lengths of major and minor axes of fit ellipse. From these values, form factor (FF; the reciprocal of circularity value) and aspect ratio (AR; major/minor) were calculated. Both FF and AR have a minimal value of 1 when a particle is a perfect circle and the values increase as the noncircle features of the particle increase. Specifically, AR is a measure of mitochondrial length and the increase of FF represents the increase of mitochondrial length and branching. This procedure is similar to mitochondrial morphology analysis as previously described [30, 32].

3. Results

3.1. Chronic EtOH Exposure Induced Mitochondrial ROS Generation.

CM-[H.sub.2]DCFDA is oxidized to DCF (dichlorofluorescein) by cellular ROS [33] and ethanol increased DCF fluorescence (images in Figure 1(a)). MitoSOX Red, which selectively targets mitochondria and is rapidly oxidized by superoxide, was used to monitor mitochondrial superoxide production (images in Figure 1(a)). In MH-S cells, five days of EtOH exposure increased both cellular ROS and mitochondrial superoxide production by ~ 100% and 50%, respectively (Figures 1(b) and 1(c)). However, EtOH-induced upregulation of cellular ROS and mitochondrial superoxide in MH-S cells were reversed by treatment with the mitochondrial targeted antioxidant, mitoT. To determine whether chronic EtOH ingestion induced ROS generation in vivo, mAMs were isolated from control or EtOH-fed mice and stained with MitoSOX and CM-[H.sub.2]DCFDA before flow cytometry or confocal imaging. Chronic ethanol ingestion upregulated cellular ROS in mAMs by ~2-fold and mitochondrial superoxide by ~3-fold (Figures 2(a) and 2(b)). Similar to that observed with MH-S cells, 24 h in vitro treatments of the mAMs with mitoT reversed EtOH-induced cellular and mitochondrial ROS production.

[FIGURE 1 OMITTED]

3.2. Chronic EtOH Exposure Resulted in Mitochondrial Dysfunction.

Mitochondrial membrane potential is a key indicator of mitochondrial function and integrity. It can be determined with TMRE, a cell permeable cationic dye that readily accumulates in active mitochondria because of the relative negative charge of the mitochondrial membrane potential. EtOH exposure resulted in two mitochondrial populations with TMRE staining. The population with higher TMRE intensity represents those with polarized mitochondria and greater capacity to transport the fluorophore. The population with lower TMRE intensity represents the depolarized mitochondria and decreased capacity to transport the fluorophore. In mAMs, chronic EtOH ingestion decreased the population of cells with higher TMRE staining by 10% (Figures 3(a) and 3(b)) suggesting loss of mitochondrial membrane potential. Similarly, EtOH exposure of MH-S cells incrementally decreased the percentage of cells with higher TMRE staining relative to the period of EtOH exposure (Figure 4(a)). In addition, in vitro and in vivo alcohol exposure also decreased the ratio of [NAD.sup.+]/NADH (Figures 4(c) and 3(c)). Since NADH is oxidized to [NAD.sup.+]+ in the process of transferring electrons in the mitochondrial electron transfer chain, decreases in the [NAD.sup.+]/NADH ratio indicate a mitochondrial redox imbalance and loss of mitochondrial function. Indeed, this EtOH-induced loss of mitochondrial membrane integrity and perturbations in the [NAD.sup.+]/NADH ratio were accompanied by a ~25% decrease in ATP production (Figure 4(b)). Ethanol-induced decreases in ATP and [NAD.sup.+]/NADH levels were both normalized through the addition of mitoT (Figures 4(b) and 4(c)).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

3.3. Chronic EtOH Exposure Induced Mitochondria Condensation and Perinuclear Clustering.

Mitochondria are dynamic organelles which constantly change their size and shape by fusion and fission and their morphological dynamics are linked to the regulation of normal cell physiology and disease. We next examined whether the EtOH-induced mitochondrial ROS generation and mitochondrial depolarization were linked to changes in mitochondrial morphology. In control MH-S, the mitochondrial network was spread throughout the cell (Figure 5(a)). With 5 consecutive days of EtOH exposure (0.2%), the majority of MH-S cells had mitochondria that were condensed and located in the perinuclear region (Figure 5(a)). To quantitatively address mitochondrial morphology changes, we analyzed mitochondrial morphology using a computer-assisted morphometric analysis, which calculates form factor (FF) and aspect ratio (AR) as discussed above. With a minimal value of 1 representing a perfect circle (major axis = minor axis), the mitochondria within control MH-S cells had AR values distributed above 4 suggesting that the mitochondria were elongated. With EtOH exposure, the majority of the AR values were below 4, suggesting a transition from an elongated shape to a more spherical shape. In addition, mitochondrial areas exceeded 15 [micro][m.sup.2] in the EtOH treatment group suggesting mitochondrial clustering. We next investigated the mitochondrial morphology after chronic EtOH ingestion. Figure 5(b) is comprised of representative confocal microscopic images of AMs isolated from control and EtOH-fed mice. Because primary mAM cells were taken from their original environment in mouse lungs, they are more fragile, and their morphology was not well retained like that for the MH-S cell line. However, the mitochondria in the AMs from the EtOH-fed mice were more fragmented and clustered at the perinuclear area when compared to the AMs from the control mice.

3.4. EtOH-Induced Impairment of Macrophage Phagocytosis Was Reversed by mitoT.

As demonstrated previously [9, 10], EtOH exposure decreased the phagocytic capacity of mAMs and MH-S cells. Since mitoT attenuated cellular and mitochondrial ROS, we next examined whether mitoT would reverse the effects of ethanol on phagocytosis. As demonstrated in our previous studies, EtOH exposure suppressed phagocytosis of the S. aureus bioparticle conjugates by 30% (Figure 6). Treatment with mitoT during the last 24 h of EtOH exposure restored the phagocytic ability of MH-S cells suggesting that mitochondrial-derived oxidative stress was central to EtOH-induced disruptions in phagocytosis. We also examined whether mitoT could restore phagocytosis to mAMs from ethanol-fed mice. Similar to that observed with MH-S cells, in vitro treatments with mitoT restored phagocytosis to the mAMs suppressed by chronic ethanol ingestion (Figure 7). These results further confirmed the association between EtOH-induced mitochondrial oxidative stress and impaired mAM phagocytosis.

[FIGURE 5 OMITTED]

3.5. EtOH Induced Early Apoptosis but Was Prevented by mitoT Treatment.

As demonstrated previously [9, 10], chronic ethanol ingestion increases AM apoptosis 3-fold with ~30% of the cells expressing markers of apoptosis. In MH-S cells with 5 days of ethanol exposure, the percentage of cells with an early marker of apoptosis, Annexin V positive staining, increased 5-fold (Figures 8(a)-8(c)) when compared to the control group. For cells positive for Annexin V plus loss of cytoplasm, ethanol increased the percentage of cells positive for late apoptosis but statistical significance was not achieved. There also was no statistically significant increase in the percentage of cells positive for a marker of necrosis, propidium iodide staining of DNA (Figure 8(c)). When MH-S cells were pretreated with mitoT, ethanol-induced early apoptosis, Annexin V positive staining, was attenuated (Figure 8(d)).

[FIGURE 6 OMITTED]

4. Discussion

Chronic alcohol abuse is associated with an increased risk of respiratory infections, pneumonia, and tuberculosis, even in those without a clinical diagnosis of an alcohol use disorder [1, 3]. However, the underlying mechanisms by which alcohol abuse increases the risk of respiratory infections are unclear. One central effect of chronic alcohol ingestion is severe oxidative stress and depletion of critical antioxidants [7]. Within the alveolar space, GSH levels in the alcoholic subjects were significantly decreased when compared with those of nonalcoholic subjects. In the alveolar epithelial lining fluid, alcohol abuse caused an -40 mV change in the glutathione and glutathione disulfide (GSH/GSSG) redox potential (Eh) [6, 7, 34]. Changes in the extracellular GSH/GSSG redox status were echoed in the intracellular GSH/GSSG redox balance of alveolar type II cells. Indeed, chronic alcohol ingestion caused a 60% decrease in GSH and induced GSH/GSSG oxidation by 40 mV in alveolar type II cells from ethanol-fed adult male rats [35]. In the mitochondria of type II cells, chronic alcohol ingestion also induced a 60 mV oxidation of the GSH/GSSG redox potential when compared to the cells from control rats [35]. For AMs, the GSH/GSSG redox state was oxidized by -30 mV after chronic ethanol ingestion [36]. Across intracellular and extracellular GSH pools in alveolar cells, the GSH/GSSG redox state was consistently oxidized by 30-60mV. AMs are the only intra-alveolar phagocyte that responds to inflammation [37] and their function is dependent on the oxidation/reduction balance in the alveolar lining fluid. Indeed, ex vivo GSH antioxidant supplementation can reverse the EtOH-induced suppression of phagocytosis in rodent models of chronic alcohol abuse [36].

Previous studies in our laboratory demonstrated that EtOH promotes oxidative stress in AMs through increased ROS production by NADPH oxidases (Nox) [10]. In that mouse model, chronic EtOH ingestion increased the level of mRNA and protein expression of Nox1, Nox2, and Nox4. Since mitochondria prodduce 90% of cellular ROS compared to a 10% cytosolic ROS contribution under baseline conditions [38], we examined the potential contribution of mitochondria to the ROS. Like the alveolar type II cells [35], the current study demonstrated that EtOH exposure (in vitro or in vivo) also upregulated mitochondrial ROS generation. Under baseline conditions, increased mitochondrial superoxide production can be enzymatically dismutated to hydrogen peroxide which is subsequently removed by catalase or glutathione peroxidase. However, chronic increases in mitochondrial ROS generation can overwhelm normal scavenging mechanisms, promote the uncoupling of the respiratory chain, and result in more ROS generation, loss of mitochondrial membrane potential, and decreased ATP. In the current study, a role for ethanol-induced mitochondrial ROS was further demonstrated by treatment of AM cells with mitoTEMPOL (mitoT) which reversed EtOH-induced mitochondrial ROS. MitoT contains a lipophilic triphenylphosphonium cation added to the TEMPOL antioxidant moiety which promotes its accumulation in the mitochondria. The TEMPOL moiety is a piperidine nitroxide, which has been widely used as a mitochondrial specific antioxidant for in vivo and in vitro studies. The TEMPOL moiety has super-oxide dismutase activity that promotes the detoxification of ferrous iron and prevents toxic hydroxyl radicals formation in the reaction of [H.sub.2][O.sub.2] with ferrous iron [39, 40]. Although our analysis of ROS by redox sensitive fluorophores has its limitations, treatment with mitoT blocked or reversed EtOH-induced ROS generation in the mitochondria further supporting that EtOH promoted mitochondrial ROS. Whether EtOH-induced mitochondrial ROS is due to interference with mitochondrial redox balances [41] or other mechanisms remains to be determined. Furthermore, mitoT also blocked the ethanol-induced increases in cytosolic ROS suggesting that mitochondrial ROS contributes to the generation of cytosolic ROS. Additional studies are needed to determine whether ROS generation through NADPH oxidases, CYP2E1, or other ROS generators are dependent on mitochondrial ROS.

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

EtOH-induced mitochondrial ROS was also associated with mitochondrial dysfunction. Mitochondrial integrity and function were interrupted by EtOH as evidenced by decreased mitochondrial membrane potential in MH-S cells. More importantly, mAMs from EtOH-fed mice displayed decreased mitochondrial function as evidenced by decreased mitochondrial membrane potential and ATP. The ratio of [NAD.sup.+]/NADH was also decreased in both the in vitro and in vivo models of ethanol exposure. Although the isolation protocol may have increased [NAD.sup.+] and NADH leak from the mitochondria, it is unlikely that the leak of one component would be preferential over the other. Since the actual leak may differ between mitochondrial preparations, we decided that expressing the concentrations of the two components as a ratio would be more accurate. In these studies, NAD+ was 0.33 [+ or -] 0.03 a.u. (absolute unit) and NADH was 0.20 [+ or -] 0.01 a.u. for the control group. For the ethanol group, NAD+ was 0.25 [+ or -] 0.03 a.u. and NADH was 0.76 [+ or -] 0.20 a.u. Therefore, the decrease in [NAD.sup.+] and increase in NADH resulted in a decrease in the mitochondrial [NAD.sup.+]/NADH ratio suggesting its oxidation in the mitochondria.

Mitochondria are organelles which supply energy for normal cellular functions making it a key regulator of cell function. For the AM, the energy intensive cellular process such as phagocytosis is particularly dependent on the capacity of the mitochondria to generate ATP. In the current studies, EtOH exposure promoted significant mitochondrial morphological changes, a central indicator of the organelle's integrity and function. In the control group, there was a network of mitochondria with an elongated shape. With EtOH exposure, the mitochondria became more spherical in shape and were present in condensed perinuclear clusters. These EtOH-induced mitochondrial morphological changes are generally associated with cellular oxidative stress and are associated with cell death [35]. As with ethanol-induced mitochondrial ROS, treatment with mitoT normalized mitochondrial [NAD.sup.+]/NADH as well as the ATP pool after ethanol exposure. This further supports a causative role for ethanol-induced mitochondrial ROS in the corresponding mitochondrial dysfunction. As observed in previous studies with ethanol-fed animals [36], there was impaired phagocytosis, a key immune function of AMs. However, in vitro mitoT treatments reversed the injurious effects of EtOH on the mitochondria and restored the phagocytic capacity even in mAMs from mice fed ethanol for 12 weeks. In addition to impaired bacterial clearance, ethanol decreased cell viability as evidenced by increased early markers of apoptosis. The capacity of mitoT treatment to block apoptosis suggested a central role for ethanol-induced mitochondrial dysfunction in the apoptotic process.

Since mitoT decreased ethanol-induced mitochondrial-and cytosolic-derived ROS, one potential mechanism for the beneficial effects of mitoT could be through its positive cytosolic effects. In previous studies, we demonstrated that chronic EtOH ingestion increases the production of TGF-[beta] and IL-13 in AMs which subsequently promotes alternative activation (M2 activation) [7]. In that study, TGF-[beta] and IL-13 activated an autocrine loop that was central to AM alternative activation. EtOH-induced upregulation of TGF-[beta] expression also promoted another self-activating autocrine loop with the constitutively active NOX 4 resulting in chronic cytosolic ROS generation [10]. Additional studies are needed to determine whether the ethanol-induced activation of TGF-[beta]/IL-13 and the TGF[beta]/NOX 4 autocrine loops are causative or secondary to ethanol-induced mitochondrial dysfunction. Alternatively, the primary driver could be through ethanol-induced mitochondrial ROS that overwhelm the large antioxidant capacity of the mitochondria, leak into the cytosol, and activate various mechanisms for cytosolic ROS such as the TGF-[beta]/NOX4 autocrine loop. In the current study, mitoT attenuated ethanol-induced mitochondrial dysfunction such as ROS, decreased ATP levels, and decreased NAD+/NADH. The mechanisms by which mitoT maintains these events that are critical for the highly energy-dependent processes of phagocytosis and maintenance of cell viability are unclear but may be through maintenance of mitochondrial GSH/GSSG, a critical event for type II cells [35].

Although each piece of data is not singularly definitive, the collective data from diverse measures indicated that ethanol increased mitochondrial ROS: (1) increased cellular ROS as indicated by CM-[H.sub.2]DCFDA oxidation which was blocked by the mitochondrial specific antioxidant mitoT; (2) increased mitochondrial ROS as indicated by MitoSOX fluorescence which was also blocked by mitoT; (3) increased cellular and mitochondrial ROS with in vitro and in vivo EtOH exposure; (4) the ability of mitoT to attenuate cellular and mitochondrial ROS in the AMs even after chronic EtOH ingestion; and (5) increased oxidation of the mitochondrial [NAD.sup.+]/NADH ratio. This mitochondrial oxidation was also associated with mitochondrial dysfunction as evidenced by loss of mitochondrial morphology, depolarization of the mitochondrial membrane potential, and decreased ATP generation. The mitochondria-targeted antioxidant mitoT not only reversed EtOH-induced mitochondrial and cytosolic ROS generation, it also reversed EtOH-induced mitochondrial dysfunction, restored AM phagocytosis, and maintained cell viability. Phagocytosis and cell viability are both complex cellular processes that are controlled at multiple points; additional studies are needed to determine the actual roles of mitochondrial ROS in EtOH-induced disruption of these cellular events. Chronically, alcohol can dampen the inflammatory responses of alveolar macrophages and the chronic suppression of phagocytosis decreases the capacity of alveolar macrophages to clear microbes. Therefore, EtOH-induced mitochondrial ROS and dysfunction in AMs may be pivotal in the increased risk of respiratory infections and ARDS in subjects with an alcohol use disorder.

http://dx.doi.org/10.1155/2014/371593

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was supported by a NIAAA T32 Training Grant (5T32AA013528-08), the Emory Alcohol and Lung Biology Center (1P50AA135757), and NIAAA (R01 AA12197). The authors acknowledge the facilities and the scientific and technical assistance of the Flow Cytometry Core Facility and Imaging Core Facility at Emory + Children's Pediatrics Research Center.

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