Prostate cancer (PCa) is the most common non-cutaneous male cancer in most populations.¹ Although the etiology of PCa is unknown, several risk factors including diet (calcium and some other nutrients) have been well identified.^{2, 3} It is also reported that the risk of having PCa drastically increase with age, being three orders of magnitude higher for the age group 40–79 years than for those younger than 39 years.^{3, 4}

Chemical elements have essential physiological functions such as maintenance and regulation of cell function, gene regulation, activation or inhibition of enzymatic reactions, and regulation of membrane function. Essential or toxic (mutagenic, carcinogenic) properties of chemical elements depend on tissue-specific need or tolerance, respectively.⁵ Excessive accumulation or an imbalance of the chemical elements may disturb the cell functions and may result in cellular degeneration or death.^{5, 6, 7}

High intraprostatic calcium (Ca) concentrations are probably one of the main factors acting in both initiation and promotion stages of prostate carcinogenesis.^{8, 9, 10} A significant tendency of age-related increase in Ca, magnesium (Mg), zinc, and many other chemical element mass fraction in the normal prostate was recently demonstrated by us. ^{9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19} Moreover, it was found that the prostatic tissue content of Ca, potassium (K), and Mg are an androgen dependent chemical element, but content of bromine (Br), manganese (Mn), and sodium (Na) are not bound to levels of sex hormone.²⁰ Thus, it seems fair to suppose that besides Ca, many other chemical elements also play a role in the pathophysiology of the prostate.

The chemical element contents in tissue of the normal^{21, 22, 23, 24, 25, 26, 27, 28} and cancerous^{29, 30, 31, 32, 33, 34} prostate have been studied, producing contradictory results. The majority of these data are based on measurements of processed tissue and in many studies tissue samples are ashed before analysis. In other cases, prostate samples are treated with solvents (distilled water, ethanol etc) and then are dried at a high temperature for many hours. There is evidence that certain quantities of chemical elements are lost as a result of such treatment.^{35, 36, 37} Moreover, only a few of these studies employed quality control using certified reference materials for determination of the chemical element mass fractions. Thus, the questions about the differences between chemical element contents in normal and cancerous prostate tissue remained open.

This work had two aims. The first aim was to obtain reliable values for the Br, Ca, K, Mg, Mn, and Na contents in the prostate cancer and in the normal prostate gland using non-destructive instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR). The second aim was to compare the levels of chemical elements in normal and cancerous tissue.

All patients suffered from adenocarcinoma of prostate (n=10, mean age M±SD was 65±10 years, range 51-77) were hospitalized in the Urological Department of the Medical Radiological Research Centre. Transrectal puncture biopsy of suspicious indurated regions of the prostate was performed for every patient, to permit morphological study of prostatic tissue at these sites and to estimate their chemical element contents. In all cases the diagnosis adenocarcinoma has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. All samples of cancerous tissue were obtained before hormonal or radiation therapy.

Normal prostates for the control group samples were removed at necropsy from 37 men (mean age 55±11 years, range 41-87), who had died suddenly. The majority of deaths were due to trauma. A histological examination in the control group was used to control the age norm conformity, as well as to confirm the absence of microadenomatosis and latent cancer.

All tissue samples were divided into two portions. One was used for morphological study while the other was intended for chemical element analysis. After the samples intended for chemical element analysis were weighed, they were freeze-dried and homogenized. The sample weighing about 10 mg (for biopsy materials) and 50-100 mg (for resected materials) was used for chemical element measurement by INAA-SLR. The samples for INAA-SLR were sealed separately in thin polyethylene films washed beforehand with acetone and rectified alcohol. The sealed samples were placed in labeled polyethylene ampoules.

To determine the contents of the chemical elements by comparison with known data for standard, aliquots of commercial, chemically pure compounds, synthetic and natural reference materials were used.³⁸ Ten sub-samples of international certified reference material (CRM) IAEA H-4 (animal muscle) weighing about 100 mg were treated and analyzed under exactly the same conditions as the prostate samples, to allow estimation of the precision and accuracy of results.

The content of Br, Ca, K, Mg, Mn, and Na were determined by INAA-SLR using a horizontal channel equipped with the pneumatic rabbit system of the WWR-c research nuclear reactor. The neutron flux in the channel was 1.7 × 10¹³n cm⁻² s⁻¹. Ampoules with prostate samples, biological synthetic standards, intralaboratory-made standards, and certified reference material were put into polyethylene rabbits and then irradiated separately for 180 s. Copper foils were used to assess neutron flux.

The measurement of each sample was made twice, 1 min and 120 min after irradiation. The duration of the first and second measurements was 10 min and 20 min, respectively. A coaxial 98-cm3 Ge (Li) detector and a spectrometric unit (NUC 8100), including a PC-coupled multichannel analyzer, were used for measurements. The spectrometric unit provided 2.9-keV resolution at the ⁶⁰Co 1,332-keV line. Details of used nuclear reactions, radionuclides, and gamma-energies were presented in our earlier publication concerning the INAA-SLR chemical element contents in human scalp hair.³⁹

All prostate samples for INAA-SLR were prepared in duplicate and mean values of chemical element contents were used in final calculation. Using the Microsoft Office Excel software, the summary of statistics, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels were calculated for chemical element contents in normal and cancerous prostate tissues. The reliability of difference in the results between the two groups of prostate tissue samples was evaluated by Student’s t-test. For the estimation of the Pearson correlation coefficient between different pairs of the chemical element mass fractions in the normal and cancerous prostate tissues the Microsoft Office Excel software was also used.

Table 1 depicts our data for Br, Ca, K, Mg, Mn, and Na mass fractions in ten sub-samples of CRM IAEA H-4 (animal muscle) certified reference material and the certified values of this material.

Table 2 presents certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the Br, Ca, K, Mg, Mn, and Na contents in normal prostate tissue and adenocarcinoma of prostate.

The ratios of means and the reliability of difference between mean values of Br, Ca, K, Mg, Mn, and Na contents in normal and cancerous prostate tissue are presented in Table 3.

Table 4 depicts results of inter-element correlation calculations (values of r – coefficient of correlation) including all pair of chemical elements identified in normal prostate tissue and adenocarcinoma of prostate.

The comparison of our results with published data for Br, Ca, K, Mg, Mn, and Na contents in normal and cancerous prostate tissue is shown in Table 5.

(n)b - number of samples.

As was shown by us the use of CRM IAEA H-4 as a certified reference material for the analysis of chemical elements contents in samples of prostate tissue can be seen as quite acceptable.⁹ Good agreement of the Br, Ca, K, Mg, Mn, and Na mass fractions analyzed by INAA-SLR with the certified data of CRM IAEA (Table 1) indicates an acceptable accuracy of the results obtained in the study of chemical elements of the human prostatic adenocarcinoma presented in Tables 2–5.

The mean values and all selected statistical parameters were calculated for 6 chemical elements (Br, Ca, K, Mg, Mn, and Na) (Table 2). The mass fraction of these elements were measured in all, or a major portion of normal and cancerous prostate samples.

From Table 2 and Table 3, it is observed that in adenocarcinoma the mass fractions of Br (p<0.0000016) and Mn (p<0.026) are significantly higher while the mass fractions of Ca (p<0.0000000038), K (p<0.022), Mg (p<0.0000042), and Na (p<0.010) are significantly lower than in normal tissues of the prostate. Thus, the mass fractions of all chemical elements investigated in the study show significant variations in cancerous tissues when compared with normal tissues of the prostate. For example, in adenocarcinoma the mean of Mn mass fraction was almost 6 times, and the mean of Br mass fraction was approximately 4 times, greater than in normal prostate tissue (Table 3). In contrary, the Ca and Mg mass fractions were nearly 3 times, and the K and Na mass fractions were approximately 20-30%, lower in adenocarcinoma than in normal prostate tissue (Table 3).

In healthy prostate tissue a half proportion (3 out of 6) of statistically significant inter-element correlations were observed for K: positive correlation with Ca (p<0.05) and inverse correlations with Br (p<0.05), and Mg (p<0.01) (Table 4). The positive inter-element correlations of Mn mass fractions with Na (p<0.01) and inverse correlations with Ca (p<0.01) mass fractions were also found in healthy prostate tissue. Additionally, levels of Mg have significant relationships with Na (p<0.01. These correlations indicate a possible connection between these elements and the normal function of the prostate. Inter-element correlations between chemical elements contents differ markedly in adenocarcinoma of prostate as compared to their relationships in normal prostate tissue. Only K contents have positive correlation with Na (p<0.001). Published data referring to inter-element correlations of Br, Ca, K, Mg, Mn, and Na mass fractions in normal and cancerous tissues of the human prostate gland were not found.

The results for all chemical element contents in the prostates of the control group (mean age 55±11 years, range 41-87) are in accordance with our earlier findings in prostates of apparently healthy men aged 41-60.⁹ In data from the literature a number of values for chemical element mass fractions in prostate tissue were not expressed on a dry mass basis. Therefore, we calculated these values using published data for water - 80% and ash - 1% on wet mass basis contents in the prostate of adult men.^{40, 41} This reported data also includes samples obtained from patients who died from different non-urological diseases. Values obtained for Br, Ca, K, Mg, Mn, and Na contents agree well with median of mean values cited by other researches for the human prostate (Table 5). In the adenocarcinoma of prostate our results were only comparable with published data for Mn and Na contents. Mean mass fractions of Ca and Mg found in this study were approximately 3-4 times lower than the means of reported data, while mean value of Br almost two orders of magnitude higher (Table 5).

Characteristically, elevated or deficient levels of chemical elements observed in cancerous tissues are discussed in terms of their potential role in the initiation, promotion, or inhibition of prostate cancer. In our opinion, abnormal levels of chemical elements in adenocarcinoma could be the consequence of malignant transformation. Compared to other soft tissues, the human prostate has higher levels of Ca, K, Mg, and some other chemical elements.^{12, 42} These data suggests that these elements could be involved in functional features of prostate tissue. Moreover, it is plausible that the reason for the emergence and development of adenocarcinoma is associated with abnormally high concentration of Ca and Mg in the prostate tissue of older men. Malignant transformation is accompanied by a loss of tissue-specific functional features, which leads to a significant reduction in the contents of such chemical elements as Ca, K, and Mg associated with functional characteristics of the human prostate tissue.

On the other hand, the well documented fact that the cancer cells, including adenocarcinoma of human prostate, are under high levels of oxidative stress.⁴³ The cancer cells exposed to oxidative stress tend to forced adaptation mechanisms including production higher levels of antioxidant enzymes such as manganese-containing superoxide dismutase (Mn-SOD). Mn-SOD has been shown to be high in human tumors including lung cancer⁴⁴, ovarian carcinoma^{45, 46}, thyroid tumours⁴⁷, renal cell carcinoma⁴⁸, brain tumors⁴⁹, esophageal and gastric cancers^{50, 51}, malignant mesothelioma^{52, 53}, hepatocellular carcinoma⁵⁴, colorectal tumors⁵⁵, breast cancer⁵⁶, and some other tumors⁵⁷ as compared to corresponding non-malignant control tissues. It was reported also that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity.⁵⁸ In cited study was shown that the human mesothelioma cells contained an extremely high level of Mn, an amount 7.3-fold higher than that in the human mesothelial cells.⁵⁸ Such great difference between Mn content in normal and malignant cells agrees well with our result for Mn mass fractions in normal and cancerous prostate tissue (Table 3).

Bromide compounds, especially potassium bromid (KBr), sodium bromide (NaBr), and ammonium bromide (NH4Br), are frequently used as sedatives in Russia.⁵⁹ It may be the reason for elevated levels of Br in specimens of patients with adenocarcinoma of prostate glad.

Our findings show that mass fraction of Ca, K, Mg, and Mn are significantly different in most adenocarcinomas as compared to normal prostate tissues (Table 3). Thus, it is plausible to assume that levels of these chemical elements in prostate tissue can be used as tumor markers. However, this subjects needs in additional studies.

In this work, chemical elemental analysis was carried out in the tissue samples of normal prostate and adenocarcinoma of prostate using INAA-SLR. It was shown that INAA-SLR is an adequate analytical tool for the non-destructive determination of Br, Ca, K, Mg, Mn, and Na content in the tissue samples of human prostate, including needle-biopsy cores. It was observed that in cancerous tissues contents of Ca, K, Mg, and Na were significantly lower and those of Br and Mn were significantly higher than in normal tissues. In our opinion, the abnormal decrease in levels of Ca, K, and Mg, as well as the increase in level of Mn in cancerous tissue could be a consequence of malignant transformation. It was supposed that elevated level of Mn as well as decreased levels of Ca, K, and Mg in prostatic tissue can be used as tumor markers.

The study was approved by the Ethical Committee of the Medical Radiological Research Center, Obninsk.

The authors are grateful to Dr Tatyana Sviridova, Medical Radiological Research Center, Obninsk, and Prof. A.A. Zhavoronkov, Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow, for supplying prostate samples.