ABSTRACT
The research is aimed at investigating the effect of sodium citrate and reduced glutathione (GSH) on arginase from human fibriod tissue.
The research shows that arginase enzyme which was partially purified was found to have a specific interaction with its substrate.
The study shows that varying the substrate concentration at constant volume of Sodium citrate and Reduced glutathione (GSH) resulted into an Un-competitive inhibition and Competitive inhibition of enzymes respectively.
The results obtained from this study shows that the inhibition nature of arginase by different inhibitors of enzymes gives a promising approach to arbnormal arginase activities in cell proliferation of uterine muscle which leads to fibroid.
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 FIBROID
Fibroid are lumps that grow on the inside and outside walls of a woman’s uterus. They are considered the most common pelvic tumor which affects one in five women of childbearing age (Galvin et al., 2010).A fibroid is a leiomyoma (benign (non-cancerous) tumor from smooth muscle tissue) that originates from the smooth muscle layer (myometrium) of the uterus (Cucinella et al., 2011). Fibroids are often multiple and if the uterus contains too many leiomyomata to count, it is referred to as diffuse uterine leiomyomatosis. The malignant version of a fibroid is extremely uncommon and termed a leiomyosarcoma. Other common names are uterine leiomyoma, myoma, fibromyoma, fibroleiomyoma (Boynton-Jarrettn et al., 2005). Fibroids are the most common benign tumors in females and typically found during the middle and later reproductive years. While most fibroids are asymptomatic, they can grow and cause heavy and painful menstruation, painful sexual intercourse, and urinary frequency and urgency. Some fibroids may interfere with pregnancy although this appears to be very rare.
Figure 1 – UTERINE FIBROID
SOURCE: WHO, 2010
The symptoms caused by uterine fibroids are a very frequent indication for hysterectomy. The effects of fibroids have been known to cause infertility and miscarriages in women, resulting in both physical and emotional distress (Massart et al., 2003). The main method of getting rid of fibroids are medical procedures, however some seems to think the holistic approach is better. A mind over matter approach seems to be highly recommended for treating fibroids naturally (WHO, 2009). Fibroids tend to be more common in African American women than in women of other racial groups. Studies have shown that fibroids are three times more likely to occur in African American women than in Caucasian women (Cucinella et al., 2011). African American women are not only at a greater risk of developing fibroid tumors, their growths tend to be larger. According to one study, an estimated 80% of African American women are affected by fibroid tumors by age 60. It is also estimated that these tumors cause symptoms in half of all African American women by age 50, compared to 30% in Caucasian women (Cucinella et al., 2011). Uterine fibroids may occur singly but most often are multiple and vary in size from an unnoticeable few millimetres to over 20 cm in diameter, significantly enlarging the abdominal cavity. They are named according to their location. Intramural fibroids lie wholly within the uterine walls, submucosal fibroids project into the uterine cavity and subserosal fibroids project from the outer surface of the uterus. They may also be pedunculated, where they are attached to the uterine wall by a stalk-like structure (Galvin et al., 2010). The aetiology and pathogenesis of fibroids are not fully understood, but their occurrence during the female reproductive lifespan indicates an association with the hormones oestrogen and progesterone. The prevalence of clinically significant fibroids peaks in the peri-menopausal years, declining after the menopause. Known risk factors for fibroids include early menarche, nulliparity, later reproductive years, obesity, Afro- Caribbean ethnic origin and tamoxifen. Fibroids appear to be 2–3-fold more common in first-degree relatives of women with fibroids, compared with the general population (Massart et al., 2003).
1.1.1 LOCATION AND CLASSIFICATION OF FIBROID
Growth and location are the main factors that determine if a fibroid leads to symptoms and problems. A small lesion can be symptomatic if located within the uterine cavity while a large lesion on the outside of the uterus may go unnoticed (Isobe et al., 2007). Different locations are classified as follows:
Intramural fibroids are located within the wall of the uterus and are the most common type; unless large, they may be asymptomatic. Intramural fibroids begin as small nodules in the muscular wall of the uterus. With time, intramural fibroids may expand inwards, causing distortion and elongation of the uterine cavity (Cesen-Cummings et al., 2003).
Subserosal fibroids are located underneath the mucosal (peritoneal) surface of the uterus and can become very large. They can also grow out in a papillary manner to become pedunculated fibroids. These pedunculated growths can actually detach from the uterus to become a parasitic leiomyoma (Isobe et al., 2007).
Submucosal fibroids are located in the muscle beneath the endometrium of the uterus and distort the uterine cavity; even small lesion in this location may lead to bleeding and infertility. A pedunculated lesion within the cavity is termed an intracavitary fibroid and can be passed through the cervix (Isobe et al., 2007).
Cervical fibroids are located in the wall of the cervix (neck of the uterus). Rarely fibroids are found in the supporting structures (round ligament, broad ligament, or uterosacral ligament) of the uterus that also contain smooth muscle tissue (Kumar et al., 2008).
Fibroid may be single or multiple. Most fibroids start in an intramural location, that is the layer of the muscle of the uterus. With further growth, some lesions may develop towards the outside of the uterus or towards the internal cavity. Secondary changes that may develop within fibroids are hemorrhage, necrosis, calcification, and cystic changes (Cesen-Cummings et al., 2003). Fibroids of uterine origin located in other parts of the body, sometimes also called parasitic myomas have been historically extremely rare, but are now diagnosed with increasing frequency (Isobe et al., 2007). They may be related or identical to metastasizing leiomyoma. They are in most cases still hormone dependent but may cause life threatening complications when they appear in distant organs. Some sources suggest that a substantial share of the cases may be late complications of surgeries such as myomectomy or hysterectomy (Kumar et al., 2008). Particularly laparoscopic myomectomy using a morcellator has been associated with a substantially increased risk of this complication.
1.1.2 PATHOGENESIS AND PATHOPHYSIOLOGY
1.1.2.1 PATHOGENESIS
Fibroids are monoclonal tumors and approximately 40 to 50% show karyotypically detectable chromosomal abnormalities. When multiple fibroids are present they frequently have unrelated genetic defects (Cesen-Cummings et al., 2003). Specific mutations of the MED12 protein have been noted in 70 percent of fibroids. Exact aetiology is not clearly understood, but the current working hypothesis is that genetic predispositions, prenatal hormone exposure and the effects of hormones, growth factors and xenoestrogens cause fibroid growth. Known risk factors are African descent, nulliparity, obesity, polycystic ovary syndrome, diabetes and hypertension (Makinen et al., 2011). Fibroid growth is strongly dependent on estrogen and progesterone. Although both estrogen and progesterone are usually regarded as growth-promoting they will also cause growth restriction in some circumstances. Paradoxically, fibroids rarely grow during pregnancy despite very high steroid hormone levels and pregnancy appears to exert a certain protective effect. This protective effect might be partially mediated by an interaction between estrogen and the oxytocin receptor.
It is believed that estrogen and progesterone have a mitogenic effect on leiomyoma cells and also act by influencing (directly and indirectly) a large number of growth factors, cytokines and apoptotic factors as well as other hormones (Mikami et al., 1998). Furthermore, the actions of estrogen and progesterone are modulated by the cross-talk between estrogen, progesterone and prolactin signalling which controls the expression of the respective nuclear receptors. It is believed that estrogen promotes growth by up-regulating IGF-1, EGFR, TGF-beta1, TGF-beta3 and PDGF, and promotes aberrant survival of leiomyoma cells by down-regulating p53, increasing expression of the anti-apoptotic factor PCP4 and antagonizing (Maruo et al., 2004) PPAR-gamma signalling. Progesterone is thought to promote the growth of leiomyoma through up-regulating EGF, TGF-beta1 and TGF-beta3, and promotes survival through up-regulating Bcl-2 expression and down-regulating TNF-alpha. Progesterone is believed to counteract growth by downregulating IGF-1 Expression of transforming growth interacting factor (TGIF) is increased in leiomyoma compared with myometrium. TGIF is a potential repressor of TGF-β pathways in myometrial cells (Mikami et al., 1998). Whereas in premenopausal fibroids the ER-beta, ER-alpha and progesterone receptors are found overexpressed, in the rare postmenopausal fibroids only ER-beta was found significantly overexpressed. Most studies found that polymorphisms in ER and PR gene encodings are not correlated with incidence of fibroids in Caucasian populations however a special ER-alpha genotype was found correlated with incidence and size of fibroids. The higher prevalence of this genotype in black women may also explain the high incidence of fibroids in this group (Mikami et al., 1998).
Uterine leiomyoma was more sensitive than normal myometrium to PPAR-gamma receptor activation resulting in reduced survival and apoptosis of leiomyoma cells. The mechanism is thought to involve negative cross-talk between ER and PPAR signaling pathways. Several PPAR-gamma ligands were considered as potential treatment. PPAR-gamma agonists may also counteract leiomyoma growth by several other mechanisms of action such as TGF-beta3 expression inhibition (Nam et al., 2007).
Hypertension is significantly correlated with fibroids. Although a causal relationships is not at all clear the hypothesis has been formulated that atherosclerotic injury to uterine blood vessels and the resulting inflammatory state may play a role. Furthermore endocrine factors related to blood pressure such as angiotensin II are suspected to cause fibroid proliferation via angiotensin II type 1 receptor (Cesen-Cummings et al., 2003).
Aromatase and 17beta-hydroxysteroid dehydrogenase are aberrantly expressed in fibroids, indicating that fibroids can convert circulating androstenedione into estradiol. Similar mechanism of action has been elucidated in endometriosis and other endometrial diseases (Mikami et al., 1998). Aromatase inhibotors are currently considered for treatment, at certain doses they would completely inhibit estrogen production in the fibroid while not largely affecting ovarian production of estrogen (and thus systemic levels of it). Aromatase overexpression is particularly pronounced in Afro-American women (Trissel et al., 2007).
Recent studies suggest that this production may represent an abnormal response to ischemic and mechanical tissue stress (Wei et al., 2007). Several factors indicate significant involvement of extracellular signaling pathways such as ERK1 and ERK2, which in fibroids are prominently influenced by hormones. Paradoxically and unlike most other conditions involving significant fibrosis the Cyr61 gene has been found downregulated in fibroids (Wei et al., 2007). Cyr61 is also known for its role as tumor suppressing factor and in angiogenesis. Hence fibroids are one of the very few tumors with reduced vascular density.
1.1.2.2 PATHOPHYSIOLOGY
Leiomyomata grossly appear as round, well circumscribed (but not encapsulated), solid nodules that are white or tan, and show whorled appearance on histological section (Yen-Ping et al., 2009). The size varies, from microscopic to lesions of considerable size. Typically lesions the size of a grapefruit or bigger are felt by the patient herself through the abdominal wall (Nam et al., 2007). Microscopically, tumor cells resemble normal cells (elongated, spindle-shaped, with a cigar-shaped nucleus) and form bundles with different directions (whorled). These cells are uniform in size and shape, with scarce mitoses. There are three benign variants: bizarre (atypical); cellular; and mitotically active (Nam et al., 2007; Yen-Ping et al., 2009). The appearance of prominent nucleoli with perinucleolar halos should alert the pathologist to investigate the possibility of the extremely rare hereditary leiomyomatosis and renal cell cancer syndrome (Yen-Ping et al., 2009).
1.1.3 SIGNS, SYMPTOMS AND CAUSES OF FIBROID
Fibroids, particularly when small, may be entirely asymptomatic. Symptoms depend on the location of the lesion and its size. Important symptoms include abnormal gynecologic hemorrhage, heavy or painful periods, abdominal discomfort or bloating, painful defecation, back ache, urinary frequency or retention, and in some cases, infertility (Zhang et al., 2008). There may also be pain during intercourse, depending on the location of the fibroid. During pregnancy they may be the cause of miscarriage, bleeding, premature labor, or interference with the position of the fetus (Wallach et al., 2004). While fibroids are common, they are not a typical cause for infertility accounting for about 3% of reasons why a woman may not have a child. Typically in such cases a fibroid is located in a submucosal position and it is thought that this location may interfere with the function of the lining and the ability of the embryo to implant. Also larger fibroids may distort or block the fallopian tubes (Zhang et al., 2008).
The most common symptoms of fibroids are;
Menstrual discomfort – Periods may be abnormally heavy and last more than a week. Some women are concerned about socially embarrassing bleeding and hesitate to engage in their normal activities. Bleeding may be severe enough to cause anemia (Wallach et al., 2004).
Bleeding between periods – Submucosal fibroids are most likely to cause abnormal bleeding, but any bleeding between periods should be checked by a physician (Alhendy and Salama, 2006).
Leg, back, or pelvic pain or pressure – A fibroid increases the size of the uterus, sometimes to the size of a 4 or 5 month pregnancy. The enlarged uterus is often “lumpy” and presses on nearby structures such as the bladder and lower intestine, causing constipation or frequent urination.
Difficulty conceiving or miscarriage – Most women with fibroids do not have fertility problems, but sometimes fibroids make it more difficult to become pregnant by natural methods. Fibroids large enough to significantly change the shape of the uterine cavity can be associated with miscarriage, premature labor, and complications of labor (Cesen-Cummings et al., 2003).
Some known factors that continuously lead to the growth of fibroid are;
Hormones: Fibroids are associated with increased estrogen production. They are rare in women less than 20 years of age as well as in postmenopausal women. Estrogen levels vary with menstruation, with menopause, and with some medications (Yen-Ping et al., 2009).
Environment: Alcohol consumption and a sedentary lifestyle have been associated with fibroids. Researchers are investigating how environmental toxins affect gene activity in the uterus (Fischer et al., 2010).
BMI: Women who are overweight or obese, based on their BMI (body-mass index), have a slightly higher risk of developing fibroids.
Pregnancy: Women who have given birth seem to have a lower risk of developing uterine fibroids. Recent information indicates that pregnancy may protect against fibroids – one theory is that fibroids are lost during the uterine changes (involution) in the weeks following childbirth (Yen-Ping et al., 2009).
Ethnicity: Not only are African-American women more likely to develop fibroids, their fibroids occur at a younger age, and are often larger and more numerous than in other ethnic groups. Asian women have a lower incidence of symptomatic fibroids (Fischer et al., 2010; Garg et al., 2011).
1.2 ARGINASE
Arginase (L-Arginine amidinohydrolase) is the final enzyme in the urea cycle, is responsible for the detoxification of ammonia in mammalian liver. It hydrolyses arginine to ornithine and urea (Lowry, 1951). It is also present in kidney, brain, intestine, mammary gland, erythrocytes, and skin. In extrahepatic mammalian tissues, arginase supplies the cell with ornithine, an important metabolite in biosynthesis of glutamic acid, proline and polyamines (Chinard, 1952). Polyamines are important in regulation of cell proliferation and differentiation and may play a role in neoplastic transformation of cells. Polyamine concentrations and ornithine decarboxylase activity (which converts ornithine to putrescine, a precursor for polyamine biosynthesis), are elevated during carcinogenesis (Süer-Gökmen et al., 2001).
Arginase activity is markedly elevated in prostatic carcinoma, gastric cancer, colorectal cancer, chronic lymphocytic leukemia, non-small cell lung carcinoma and breast cancer. Increased arginase activity has also been reported in cancer metastases. Other than arginase, enzymes involved in the production of urea have not been found in human epidermal tissue (Chinard, 1952). Increased arginase activity in mouse epidermis may provide ornithine for polyamine biosynthesis and for production of glutamate and proline and of keratinous proteins (Geyer and Dabich, 1971). Epidermal arginase activity is increased in psoriasis, an inflammatory disease of the skin characterized by epidermal hyperproliferation. Most skin tumors are benign, e.g., verruca, nevi, keratosis, cysts, and skin tags, but a significant number are premalignant or malignant. A small percentage of actinic keratoses and the cellular-type lesions of blue nevi and junction nevi may undergo malignant transformation. Arginase activity and ornithine concentration are elevated in chemically-induced benign tumors in mice and it was found to be elevated in malignant tumors of the human skin (Lowry, 1951; Chinard, 1952). The metabolic fates of arginase are differentially expressed according to cell type, age and developmental stage, diet and state of health and disease.