OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION IN HUMAN IMMUNODEFICIENCY VIRUS PATIENTS ON HIGHLY ACTIVE ANTIRETROVIRAL THERAPY

ABSTRACT

This study was aimed at evaluating oxidative stress and mitochondrial dysfunction in HIV patients on a combination of emtricitabine, tenofovir and efavirenz (ATRIPLA), commonly used for treating these patients attending the HIV clinic of Enugu State University Teaching (ESUT) Hospital, Parklane, Enugu, Nigeria following short and long-term therapy. Ninety six (96) subjects (divided into four groups) aged between 18 and 60 years were recruited for the study from the HIV clinic and the laboratory of the hospital. Ethical clearance was obtained from the Ethical Committee of the same hospital.   Group 1 consisted of twenty four (24) apparently healthy HIV sero-negative age–matched individuals (No HIV or control group) who work in different laboratories of the hospital. Group 2 consisted of 24 sero-positive patients who had not started any form of treatment (treatment naive group), while group 3 was made up of twenty four (24) subjects on a short-term course of highly active antiretroviral therapy (HAART) (for less than one year). Group 4 was made up of twenty four (24) subjects who were on HAART for more than one year, representing those on long-term therapy. Ten mililiters (10 ml) of blood sample was collected from each patient from the antecubital-vein without venous stasis into EDTA and plain bottles.

Serum was processed from the retracted whole blood and stored in duplicates in cryobottles at -200C for

biochemical analyses while the anticoagulated blood samples were further processed for CD4+ cell count and DNA extraction for genomic studies. Total antioxidant capacity (TAC in nmol/l), malondialdehyde (MDA in mmol/l), lactate level (in mg/dl), creatine kinase activity (Ck-MB isoform in IU/L), triacylglycerols (TAG) concentrations in mg/dl, CD4+ count in cells/μl, alanine aminotransferase (ALT) activity in IU/L and genomic studies were all done using standard operative procedures. A comparison of the various groups showed a non-significant (p > 0.05) difference in lactate concentration across the

study groups with groups 1,  2, 3 and 4 having lactate concentrations of 10.71±0.37, 12.84 ±0.59,

10.68±0.38  and  10.20±0.18  respectively.  Group  2  subjects  had  significantly  (p  <  0.05)  higher malondialdehyde (MDA) concentration (25.33±0.38) compared to group 1 (1.63±0.35), group 3 (12.29±0.20) and group 4 (14.72±0.78) respectively. The ALT activity was significantly (p < 0.05) higher in group 2 (46.51±0.80) compared to group 1 (22.43±1.07), group 3 (31.05±1.10) and group 4 (39.93±0.92) respectively. HIV-infected patients on HAART for less than 1 year (group 3) had significantly (p < 0.05) higher total antioxidant status (TAS) at 1208.21±12.56 compared to group 1 (No HIV control  group) at 1172.67±20.42, greater than 1year on HAART at 500.88±6.13 and Naïve HIV group at 402.17±5.53 respectively. The TAS of group 3 subjects (subjects less than 1 year on HAART) was significantly (p < 0.05) higher than that of group 1 subjects (No HIV subjects), group 2 subjects (Naïve HIV subjects) and group 4 (subjects greater than 1 year on HAART).    Creatine kinase (CK) activity was significantly (p < 0.05) higher in group 2 (285.36±33.18) compared to group 1 (104.62±16.55), group 3 (178.95±13.54) and group 4 (207.75±22.40) respectively. There was a non- significant (p > 0.05) difference in triacylglycerols (TAG) concentration across the study groups with groups 1, 2, 3 and 4 having TAG concentrations of 131.25±10.54, 128.17±12.41, 124.24±9.53 and

129.13±10.63 respectively. The CD4+  count of group 1 subject (748.04±25.26) was significantly (p <

0.05) higher than that in group 2 (258.54±54.11), group 3 (422.42±30.08) and group 4 (680.84±48.41). Subjects on long term treatment of HAART had significantly (p < 0.05) higher CD4+ count compared to the treatment naïve group and those on short term treatment. For the genomic studies, the average normalized  copy  number  of  the  mitochondrial  genes  under  review  (mtDNA:  D-loop,  ATPase  8,

uur) TRNALEU were greater for the naïve and those on therapy for more than 1 year while mtDNA (ND4) showed  no  copy  number  variation  across  the  groups.  The  treatment  naïve  group  had  the  highest expression of the mt-141 gene (ATPase 8) with copy number (11.84±1.78). However, the group on HAART for more than a year had a significantly (p < 0.05) higher expression of the mt-141 gene (ATPase 8) with copy number (5.38±0.98) as well as the short-term treated group with copy number 2.45 ±0.43 compared to the No-HIV group (0.08±0.07). The present study showed that HIV infection and long-term use of HAART increase oxidative stress which may impact on the mitochondrial function.

CHAPTER ONE INTRODUCTION

Human Immunodeficiency Virus (HIV)  is  the  cause of the  fatal  condition called  acquired immunodeficiency syndrome (AIDS). AIDS was first recognized in the United States in 1980-

1981 when homosexual men were found to have unusual infections and tumours suggesting an underlying deficiency in their cell-mediated immunity (Maartens et al., 2014).  Evidence exists to show that HIV has been present in humans for 15-20 years before then but the exact origin of the virus is not yet known (CDC, 2013).

Over the years, relative decline in  morbidity and mortality of human immunodeficiency virus (HIV) infection in some countries has been observed due to the use of a potent combined therapy known as highly active antiretroviral therapies (HAART) (Maartens et al., 2014).  It has led to a decrease in viral load (a measure of the amount of HIV virus in the blood), quantitative and qualitative  improvement  of immune  functions  in patients,  especially CD4+   T-  lymphocytes count, resulting in a decrease of infectious complications and a global clinical improvement (Klatt and Silvestri, 2012).  In spite of the positive effects of HAART on the immune system and on the metabolic alterations during HIV infections, it has been reported that the commonly used drugs zidovudine (AZT), didanosine (ddI) and stavudine (d4T) are toxic to hepatocytes and other tissues (Heil et al., 2010).

Recent reports continue to point to the mitochondria as the target for toxicity. This is so because in addition to  impairing the HIV replication machinery (by inhibition of their target– HIV reverse   transcriptase),  these   drugs  also   inhibit   the   human  polymerase  called   “gamma polymerase” which is responsible for the replication of mitochondrial DNA (mtDNA) (Fraser, et al., 2014).  Decline in mtDNA leads to defects in respiratory chain function because the mtDNA encode for about 13 subunits of the respiratory chain complexes located in the inner mitochondrial membrane (Apostolova et al., 2011). This compromises oxidative synthesis of ATP and results in overdependence on cytosolic glycolysis for energy generation leading to accumulation of lactate (Maartens et al., 2014).  A normal respiratory chain function is essential for the synthesis of DNA. Mitochondrial DNA (mtDNA) depletion and thus mitochondrial

toxicity in the liver is associated with elevated liver transaminases (Koczor and Lewis, 2010). Studies have also reported a drug-induced mitochondrial toxicity with asymptomatic elevation of serum lipase and triacylglycerols and myopathy with elevated creatine kinase. These represent some biochemical parameters reported to be elevated following mitochondrial toxicity (Apostolova et al., 2011). Oxidative stress, defined as a disturbance in the equilibrium status of pro-oxidant/antioxidant systems, in favour of oxidants, involving free radicals on intact cells, is particularly an important factor in mitochondrial dysfunction (Malik and Czajka, 2013). This is because the respiratory chain leaks the superoxide free radical which may react with nitric oxide to form the damaging by-product, peroxynitrite (Koczor and Lewis, 2010).  The mitochondria is thus a major source of reactive oxygen species (ROS) leading to oxidative damage to mitochondrial proteins,  membranes, and DNA (including mtDNA); impairing the ability of mitochondria to synthesize ATP and to carry out other metabolic functions. This contributes to a wide range of pathologies (Murphy, 2009; Malik and Czajka, 2013).

Diseases of the mitochondria may be inherited or acquired; and the treatment with highly active anti-retroviral therapies may represent  an  acquired cause.  Interplay of oxidative  stress and mitochondrial dysfunction may increase mtDNA mutations. Thus, with increased use of these drugs, mtDNA mutations and dysfunction may become increasingly important, pathophysiologically (Malik and Czajka, 2013).

1.1.1 Origin of HIV

The origin of human immunodeficiency virus has remained a vexed issue beclouded with strong racial   overtones.   The   general   Western   view   is   that   a   related   virus   called   simian immunodeficiency virus (SIV) which infected the African green monkey (AGM) some years earlier crossed the species barrier into humans (Eze, 2009). This was adduced to explain the high prevalence of HIV-1 infection in Central Africa. Although SIV causes little or no adverse effects on the AGM, it is believed that the serious damaging effects of HIV are characteristic of viruses that jump species barrier (Maartens et al., 2014).

Fortunately for Africa, it is now known that SIV has less serologic cross-reactivity with HIV-1 which is the predominant infection in Central Africa than HIV-2 which is almost exclusively

found in West Africa. Thus, if the species jump hypothesis were correct; HIV infections and AIDS would have existed in West Africa earlier than in Central Africa and by extrapolation, both the infection and disease would have been much more widespread in West than in Central Africa. This is of course, not the case; since HIV and AIDS appeared much later and have maintained lower prevalence in some countries of West Africa than in Central Africa. The infection was first reported in Nigeria around 1986 (Bashorun et al., 2014).

It is fair to presume that HIV infection started in America in the 1970’s. The infection was transported to East and Central Africa by visitors to the numerous tourist attractions in those regions of Africa. This explains the predominance of HIV-1 infection there as is the case in America.  The time frames for the spread of HIV infection in both America and Central Africa are similar. West African States, with limited tourist trade (such as Nigeria) became infected later and slowly than East and Central Africa (Eze, 2009).

The true origin of the AIDS virus may not be known. However, there is scientific probability that human AIDS might have started accidentally in a certain line of cancer-research going on in numerous nations during the 1960’s. This line of reasoning is supported by accusations by both the East and the West over the responsibility for introducing HIV into the general population.  It is therefore probable that the AIDS virus originated from the laboratory rather than in Africa as speculated (Fraser et al., 2014).

1.1.2 Classification of HIV

HIV belongs to the family of viruses called retroviruses and subfamily lentiviruses. Retroviruses are single-stranded ribonucleic acid (RNA) viruses that contain the enzyme reverse transcriptase (CDC, 2010). This enzyme is an RNA-directed DNA polymerase that possesses ribonuclease activity.  It enables the RNA of the virus to replicate a deoxyribonucleic acid (DNA) copy of itself in order to become integrated and replicate in host cells (Fraser et al., 2014).  The ability to transcr  DNA from RNA is a unique feature of retroviruses and gives them their name.  Normally RNA is transcribed from DNA. HIV is the first described human lentivirus.   Most known lentiviruses infect animals, causing slow progressive diseases, including immunodeficiency in some animals.  Lentiviruses are spherical particles, measuring 100-140 nm in diameter with a

cylindrical core. Currently, two genetically and immunologically distinct human immunodeficiency viruses have been identified, characterized and recognized as causing HIV and AIDS. They are: HIV-1 and HIV-2 (Fraser et al., 2014).

HIV-1 has the widest distribution, being prevalent in every part of the world while HIV-2 is found mostly in West Africa. The existence of a third, the type O (variant of HIV type 1) has been proposed. An increasing number of different strains of both HIV-1 and HIV-2 are being identified by molecular virology and phenotyping in cell culture (Fraser et al., 2014).  Highly cytopathic and  infectious strains of HIV-1 have been identified in parts of Central Africa. Increase in virulence appears to be due to minor differences in the molecular structure of the virus. Some strains of HIV-2 appear to cause few symptoms in those known to have been infected for many years since HIV-2 presents longer period of asymptomatic status and runs a slower disease course (Fraser et al., 2014).

1.2 Structure of HIV

A complete HIV particle consists of the envelope, capsid (shell) and core

Fig. 1: Structure of the HIV Virus

Source: Engelman and Chereppanov, 2012

1.2.1 Envelope

This membrane surrounds the virus. It is a lipid bilayer derived from the host cell membrane and contains some host proteins (Van der Kuyl and Berkhout, 2012). Embedded in the envelope is the viral encoded glycoprotein (gp) gp 41. Bound to this is the outer glycoprotein knob gp 120. The knobs are used to attach the virus to its host cell. The gp 120 molecules bind to specific

molecules on the surface of the host cell called CD4+ receptors (Fraser et al., 2014).

1.2.2 Capsid

This is the protein coat that surrounds inner mass of the virus which contains two identical single strands of viral RNA, structural proteins, the enzyme reverse transcriptase and other enzymes. The main core protein is p24. The RNA carries the genetic material (genome) of the virus (Van der Kuyl and Berkhout, 2012).

1.2.3 HIV Genome

Structural Genes of HIV

There are three structural genes that encode for the structures of HIV. They are as follows:

      env (envelope) gene that encodes for glycoproteins, gp120 and gp41, which make up the envelope of the virus. The p160 which is formed in infected cells is the precursor product of the envelope gene.  When changes in the genetic make-up of HIV occur, they mainly affect the envelope (Pandit and Sinha, 2011).

      gag (group associated antigen) gene which encodes for proteins that form capsid and core of the virus.  These are the inner core protein p24, the proteins p7 and p9 which bind the RNA molecules, and p17 which forms the capsid.  The precursor product of these proteins is p55, which is present in intact virus particles (Van der Kuyl and Berkhout, 2012).

      pol  (polymerase)  gene  which  encodes  for  the  viral  enzymes.  These  are  the  reverse transcriptase/ribonuclease p61 and p52, protease p10 and endonuclease p31. The p160 which is present in intact virus particles is the precursor product of these enzymes (Van der Kuyl and Berkhout, 2012).

Regulatory Genes of HIV

In addition to the three structural genes found in all retroviruses, HIV possesses other genes that regulate the assembly of viral proteins and control infectivity, viral replication, and latency (Pandit and Sinha, 2011).   The following are among the regulatory genes of HIV for which functions are known:

      tat (trans-activator) gene which is detected mainly in the nucleus of infected cells and is essential for viral replication.

    rev (regulatory) gene which is involved in regulating the production of viral proteins

    vif (virion infectivity factor) gene which is necessary to produce infectious particles.

      nef (negative regulatory factor) gene which slows down the transcription of the viral genome and may therefore be responsible for HIV remaining dormant in infected cells.

Note: HIV- 1 possesses a gene called VPU which is not present in HIV – 2 and   a gene called VPX is contained in HIV-2 and not in HIV-1. These genes may account for the difference in incubation times between these two viruses in some parts of West Africa.

1.3 Replication of HIV

HIV may infect any cell bearing CD4+  antigen receptor.   Such cells are mainly the helper- inducer subsets of T lymphocytes referred to as T4 lymphocytes (Van der Kuyl and Berkhout,

2012). CD4+ antigen is also found on 5-10% of B lymphocytes, 10-20% of tissue macrophages

and up to 40% of circulating monocytes. It is thought that macrophages and monocytes are important reservoirs of HIV. Monocytes are able to carry the virus to various organs in the body such as lungs and brain (Fraser et al., 2014).

1.3.1 The HIV Life Cycle

Step 1: Binding and Fusion

HIV begins its life cycle when it binds to a CD4+ receptor and two co-receptors on the surface of CD4+  T-lymphocyte.  The virus then fuses with the host cell. After fusion, the virus releases RNA, its genetic material, into the host cell (Van der Kuyl and Berkhout, 2012).

Step 2: Reverse Transcription

An HIV enzyme called reverse transcriptase converts the single-stranded HIV RNA to double- stranded HIV DNA (Van der Kuyl and Berkhout, 2012).

Step 3: Integration

The newly formed HIV DNA enters the host cell’s nucleus, where an HIV enzyme called integrase hides/integrates the HIV DNA into the host cell’s own DNA.   The integrated HIV DNA is called provirus.  The provirus may remain inactive for several years, producing few or no new copies of HIV (Pandit and Sinha, 2011).

Step 4: Transcription

When the host cell receives a signal to become active, the provirus uses a host enzyme called RNA polymerase to create copies of the HIV genomic material, as well as shorter strands of RNA called messenger RNA (mRNA). The mRNA is used as a blueprint to make long chains of HIV proteins (Pandit and Sinha, 2011).

Step 5: Assembly

An HIV enzyme called protease cuts the long chains of HIV proteins into smaller individual proteins.    As  the  smaller  HIV  proteins come together with copies of HIV’s RNA genetic material, a new virus particle is assembled (Pandit and Sinha, 2011).

Step 6: Budding

The newly assembled virus pushes out (buds) from the host cell.  During budding, the new virus steals part of the cell’s outer envelope.  This envelope which acts as a covering is studded with protein/sugar combinations called HIV glycoproteins. These HIV glycoproteins are necessary for the virus to bind CD4+  and co-receptors.  The new copies of HIV can now move on to infect other cells (Pandit and Sinha, 2011).

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