**2. Testosterone: A basal overview of biosynthesis, metabolism and its action**

In the human male, the main circulating androgen is testosterone (TST). More than 95% of circulating TST is secreted by the testis (Leydig cells) which produce aproximately 6-7 mg of TST daily (9). The rest is secreted by the adrenal cortex, and very small quantities (especially pregnan derivatives) are formed by the cells of the brain (10).

Physiologic TST level in a male is 3-8 ng / ml. The source for the synthesis of steroids is cholesterol. This substrate may be synthetized *de novo* from acetate but it may be also taken up from plasma lipoproteins. Cleavage of the side chain of cholesterol in the mitochondria and the formation of pregnenolone (biologically inactive) is the start of steroidogenic cascade. Pregnenolone is further converted into various steroids by enzymes (cytochromes P450) in the endoplasmatic reticulum.

TST secretion is regulated by the hypothalamic-pituitary-gonadal axis. The hypothalamic luteinising hormone-releasing hormone (LHRH) stimulates the anterior pituitary gland to release luteinising hormone (LH) and follicle-stimulating hormone (FSH). The main regulator of Leydig cell function is LH, acting through the LH receptor (LHr) in Leydig cells.

Androgen Deprivation Therapy for Prostate Cancer 339

Androgen metabolites are excreted as free steroids or bound (conjugated). Conjugated steroids are bound to glucuronide or sulfate group. Androgens are mostly degraded in the liver (glucuronate, sulphates), but the prostate the skin also contribute significantly to the metabolism of androgens. All the steroid - metabolising enzymes constitute a network for transforming androgens into secretion products (conjugated, unconjugated) that finally leave the body via the urine or the skin. The flux though this network is great, because the

Growth and resorption of bone tissue are mediated by *osteoblasts* and *osteoclasts*. Both types of cells exert mutual influence on each other and equilibrium between the activity of both cell lines maintains net bone mass during constant renewal and turnover. Decreased osteoblast activity and increased activity of osteoclasts leads into loss of bone mass. AR have been located on normal human osteoblasts (24) and both aromatizable and nonaromatizable androgens can stimulate of human osteoblasts proliferation in vitro (25). Bone deformation strain represents a stimulus for osteoblastic activity. Androgens modify the effects induced by the mechanoreception of human osteoblastic cells by affecting adhesion molecule expression, i.e. fibronectin and the fibronectin receptor. These substances facilitate the adhesion of bone cells to the extracellular matrix, which represents a crucial requirement for osteoblastic development and function (26). In addition, the secretion of osteoprotegerin (OPG), which is unaffected by mechanical strain alone, is doubled when this stimulus occurs in the presence of androgens. OPG is a decoy receptor for RANKL (receptor activator of nuclear factor-kappaB ligand). RANKL is secreted by osteoblasts, it induces osteoclastogenesis stimulates osteoclast differentiation (27). Thus, OPG inhibits bone resoptive effect induced by RANKL (26). Accordingly, TST levels directly correlate

Parathyroid hormone (PTH) induces osteoclast formation a differentiation. Androgens have direct inhibiting effect on this process via osteoclasts, which express AR and these cells are also blocked form PTH effects by androgens when no conversion to estrogens occurs (29). Androgens decrease the number of bone remodeling cycles by modifying the genesis of osteoclasts and osteoblasts from their respective progenitor cells. In addition, androgens also exert effects on the lifespan of mature bone cells: they exert pro apoptotic effects on osteoclasts and anti-apoptotic effects on osteoblasts and osteocytes. TST also modulates

Osteoblast activity is reflected by concentration of procollagen type 1 (carboxy-terminal: P1CP or amino-terminal: P1NP) and other non collagenous proteins secreted by osteoblasts, eg. osteocalcin and bone specific alkaline phosphatase (BSAP). Also OPG as a decoy receptor for RANKL, can serve as marker of osteoblast activity. Bone resorption, hence osteoclast activity, therefore, can be estimated by urinary excretion of degradation products of type I collagen, such as deoxypyridinoline (DPD) and collagen type I cross- linked N-

An independent role of androgens in protecting bone mass, both by promoting bone formation and attenuating bone resorption has been demonstrated in humans. Nevertheless,

Aromatization of TST to estradiol is a pivotal event concerning effects of sex steroids on bone metabolism. Estrogen receptors (ER) have been localized in human osteoblasts (32),

the role of its metabolite estradiol is pivotal in bone metabolism (30).

effects induced by other hormones and cytokines involved in bone metabolism (30).

half-life of TST in men is only 12 minutes (12).

with OPG concentrations in healthy men (28).

telopeptide (NTX) (31).

**3. Androgens and bone metabolism** 

LH and FSH are required for the development and maintance of testicular functions.The natural ligand for the LHr is LH, but also human choriogonatropin (hCG) can equally well activate the LHr. Activated LHr stimulate adenylyl cyclase via GTP binding proteins and this results in increased production of cyclic AMP (cAMP). cAMP increases steroid production (11).

The total concentration of steroids in target tissues (central and peripheral nervous system, bone, muscle, adipose tissue, haematopoetic system and myocardium) and body fluids is dependent on the presence of binding proteins (sex hormone binding globulin, SHBG, albumin). Binding proteins represent a storage form of circulating steroids, which bind 98% of circulating TST. The rest-2% is "free testosterone" (fTST), which is biologically active. Homeostasis is achieved by "closed" steroid feedback inhibition mechanism, where the plasmatic level of steroids affects the secretion of LH from adenohypophysis.

The effect of TST on target tissues is modulated by metabolic pathways.


In addition to these metabolic pathways, the level of DHT in target tissues is affected by other enzymes (hydroxysteroid dehydrogenases), which "fine-tune" the effect of androgens in the target tissues (17). Owing to these local conversions the peripheral plasma concentration of androgens are only a rough indicator for their biological activities (12).

The mechanism of action of androgens can be divided into genomic and non-genomic effect (12). Non - genomic effects of androgens include mechanisms affecting the flow of calcium in the cells and the effect on phosphorylation cascade of Map-kinase (18, 19), or membrane effects (20). Genomic effects are mediated by activation of androgen receptor (AR). AR acts as a transcription factor activated by its ligand (TST). By androgen binding, AR is translocated from the cytoplasm to the nucleus where it binds to its DNA domain and interacts as a homodimer with specific DNA sequences that are referred as androgen responsive elements (ARES) (21). Its binding to DNA leads into interactions with transcription factors (22) and other co-factor proteins (21). This results into the "up" or "down" regulation of transcription of target genes (23).

LH and FSH are required for the development and maintance of testicular functions.The natural ligand for the LHr is LH, but also human choriogonatropin (hCG) can equally well activate the LHr. Activated LHr stimulate adenylyl cyclase via GTP binding proteins and this results in increased production of cyclic AMP (cAMP). cAMP increases steroid

The total concentration of steroids in target tissues (central and peripheral nervous system, bone, muscle, adipose tissue, haematopoetic system and myocardium) and body fluids is dependent on the presence of binding proteins (sex hormone binding globulin, SHBG, albumin). Binding proteins represent a storage form of circulating steroids, which bind 98% of circulating TST. The rest-2% is "free testosterone" (fTST), which is biologically active. Homeostasis is achieved by "closed" steroid feedback inhibition mechanism, where the

1. *Aromatisation* of TST gives rise to 17b- estradiol. When the target cell is estrogendependent, the aromatase activity in target cells and supply of androgen substrate (TST) are of major importance for determining the rate of synthesis of estrogens. Aromatase cytochrome P450 enzyme is expressed in many tissues including placenta,

2. *Reduction* of TST into 5-alfa-dihydrotestosterone (DHT) is achieved by 5a-reductase. This active form of TST (5 to 10 times more biologically effective) can fully activate the androgen receptor (AR) (12). There are 3 isoforms of 5a-reductase. Isoform 2 is clinically more important because its deficiency is associated with distinct clinical manifestations (13). Isoform 2 predominates in cells of the prostate and external genitalia, while isoform 1 predominates in the cells of the skin (except genitals), and in liver cells in small amounts. However, in prostate cancer cells, overexpression of isoform 1 is a common finding, thus increasing its clinical significance (14). In the total deficiency of isoform 2 (autosomal recessive) there is a serious alteration of the development of sex organs in utero (male pseudohermafroditism). Many mutations of the gene encoding isoform 2 are known and can result into a number of different clinical manifestations. Signs of its deficiency are small phallus, severe hypospadias, scrotum bifidum, residual prostate utriculus (15). The newly discovered isoform 3 may play an important role in the development of hormone-refractory prostate cancer (HRCaP) as its overexpression

In addition to these metabolic pathways, the level of DHT in target tissues is affected by other enzymes (hydroxysteroid dehydrogenases), which "fine-tune" the effect of androgens in the target tissues (17). Owing to these local conversions the peripheral plasma concentration of androgens are only a rough indicator for their biological activities (12). The mechanism of action of androgens can be divided into genomic and non-genomic effect (12). Non - genomic effects of androgens include mechanisms affecting the flow of calcium in the cells and the effect on phosphorylation cascade of Map-kinase (18, 19), or membrane effects (20). Genomic effects are mediated by activation of androgen receptor (AR). AR acts as a transcription factor activated by its ligand (TST). By androgen binding, AR is translocated from the cytoplasm to the nucleus where it binds to its DNA domain and interacts as a homodimer with specific DNA sequences that are referred as androgen responsive elements (ARES) (21). Its binding to DNA leads into interactions with transcription factors (22) and other co-factor proteins (21). This results into the "up" or

plasmatic level of steroids affects the secretion of LH from adenohypophysis. The effect of TST on target tissues is modulated by metabolic pathways.

ovary, testis, fat tissue, liver, brain, hair follicles.

is found in the HRCaP cells (16).

"down" regulation of transcription of target genes (23).

production (11).

Androgen metabolites are excreted as free steroids or bound (conjugated). Conjugated steroids are bound to glucuronide or sulfate group. Androgens are mostly degraded in the liver (glucuronate, sulphates), but the prostate the skin also contribute significantly to the metabolism of androgens. All the steroid - metabolising enzymes constitute a network for transforming androgens into secretion products (conjugated, unconjugated) that finally leave the body via the urine or the skin. The flux though this network is great, because the half-life of TST in men is only 12 minutes (12).

#### **3. Androgens and bone metabolism**

Growth and resorption of bone tissue are mediated by *osteoblasts* and *osteoclasts*. Both types of cells exert mutual influence on each other and equilibrium between the activity of both cell lines maintains net bone mass during constant renewal and turnover. Decreased osteoblast activity and increased activity of osteoclasts leads into loss of bone mass. AR have been located on normal human osteoblasts (24) and both aromatizable and nonaromatizable androgens can stimulate of human osteoblasts proliferation in vitro (25).

Bone deformation strain represents a stimulus for osteoblastic activity. Androgens modify the effects induced by the mechanoreception of human osteoblastic cells by affecting adhesion molecule expression, i.e. fibronectin and the fibronectin receptor. These substances facilitate the adhesion of bone cells to the extracellular matrix, which represents a crucial requirement for osteoblastic development and function (26). In addition, the secretion of osteoprotegerin (OPG), which is unaffected by mechanical strain alone, is doubled when this stimulus occurs in the presence of androgens. OPG is a decoy receptor for RANKL (receptor activator of nuclear factor-kappaB ligand). RANKL is secreted by osteoblasts, it induces osteoclastogenesis stimulates osteoclast differentiation (27). Thus, OPG inhibits bone resoptive effect induced by RANKL (26). Accordingly, TST levels directly correlate with OPG concentrations in healthy men (28).

Parathyroid hormone (PTH) induces osteoclast formation a differentiation. Androgens have direct inhibiting effect on this process via osteoclasts, which express AR and these cells are also blocked form PTH effects by androgens when no conversion to estrogens occurs (29).

Androgens decrease the number of bone remodeling cycles by modifying the genesis of osteoclasts and osteoblasts from their respective progenitor cells. In addition, androgens also exert effects on the lifespan of mature bone cells: they exert pro apoptotic effects on osteoclasts and anti-apoptotic effects on osteoblasts and osteocytes. TST also modulates effects induced by other hormones and cytokines involved in bone metabolism (30).

Osteoblast activity is reflected by concentration of procollagen type 1 (carboxy-terminal: P1CP or amino-terminal: P1NP) and other non collagenous proteins secreted by osteoblasts, eg. osteocalcin and bone specific alkaline phosphatase (BSAP). Also OPG as a decoy receptor for RANKL, can serve as marker of osteoblast activity. Bone resorption, hence osteoclast activity, therefore, can be estimated by urinary excretion of degradation products of type I collagen, such as deoxypyridinoline (DPD) and collagen type I cross- linked Ntelopeptide (NTX) (31).

An independent role of androgens in protecting bone mass, both by promoting bone formation and attenuating bone resorption has been demonstrated in humans. Nevertheless, the role of its metabolite estradiol is pivotal in bone metabolism (30).

Aromatization of TST to estradiol is a pivotal event concerning effects of sex steroids on bone metabolism. Estrogen receptors (ER) have been localized in human osteoblasts (32),

Androgen Deprivation Therapy for Prostate Cancer 341

In men, osteoporosis occurs later than in women (44), but the prevalence of osteopenia does not differ significantly between men and women aged more than 50 years. Conversely, the prevalence of osteoporosis in men is lower than in women (40). Even though it may be underestimated when standard female BMD parameters are considered suitable for normal mineralization in men (45). Men generally have a higher BMD than women at the same age (46). Accordingly, the prevalence of male osteoporosis is greater when male-specific ranges are used in men above fifties: ranging from 1% to 4% of elderly men when the diagnosis is

Men are estimated to lose bone mineral density (BMD) at a rate of up to 1% per year with advancing age (47, 48), and one in eight men over age 50 years will experience an osteoporosis-related fracture in their lifetime (49). Of all osteoporotic fractures, hip fractures contribute to the greatest morbidity as well as mortality, both of which are much greater in

Current guidelines recommend assessment of bone mineral density (BMD) previous to ADT and yearly thereafter (53) with dual-energy x-ray absorptiometry (DXA) which is considered the standard method to measure BMD (54). International Society for Clinical Densitometry (ISCD) recommends that central skeleton sites (lumbar spine, total hip and femoral neck) are

Diagnosis of osteoporosis can than be made according to WHO classification: if T- score is less than 2,5 of standard deviation. Values between (-) 1 and (-) 2.5 SD (standard deviation) is defined as osteopenia. T - score stands for the number of standard deviations (SD) from

To improve the identification of patients at highest risk of fracture, WHO has developed an

According to European Association of Urology (EAU) guidelines, a precise evaluation of BMD should be performed by dual X-ray absorptiometry before starting long-term ADT. An initial low BMD (T-score below 2.5, or below 1 if other risk factors are present) indicates a high risk of subsequent non-metastatic fracture, suggesting the need for early use of preventive

> Clasiffication T-score Normal >-1 Osteopenia -1 - -2,5 Osteoporosis <-2,5

At presence, significant amount of data report that ADT is associated with the loss of BMD in a time-dependent manner (57-59) which leads into increased fracture risk (60). Skeletal fractures negatively correlate with overall survival in men with PCa (61) and

Treatment of complications of pathological fractures is complicated and expensive (63). Moreover, the typical feature of PCa is the ability to metastasize into bone in more than 80 % of cases (64). Most bone lesions in PCa are osteoblastic in nature (65). However, studies

based on female cut-off points vs. 3% to 6% when based on male cut-off points (40).

men than in women (50-52).

bisphosphonate therapy (56).

Table 1.

**5.2 Diagnosis of osteoporosis** 

the most appropriate locations to asses BMD (55).

the density of young healthy individuals of the same sex (39).(Table 1)

algorithm to predict fractures - FRAX ™ http://www.shef.ac.uk/FRAX/

**5.3 ADT, prostate cancer (PCa) and clinical aspects of bone disease** 

maintaining skeletal health is crucial for QoL and survival (62).

osteoclasts (33), and osteocytes (34). Human males with mutations of ER or aromatase genes do not achieve normal bone density, despite normal or increased levels of serum TST (35).
