2.1

Subjects

After Institutional Review Board approval (RP.20.12.88; MS.23.02.2303) and obtaining written consent, this research was conducted between October 2020 and October 2023. It included 125 BC patients who underwent RC (99 males/26 females), and 72 controls (45 males/ 27 females), with a mean age ± standard deviation (SD) 63.8 ± 8.2 and 45.7 ± 8.6 years, respectively. BC patients included 26 farmers, 10 painters, 25 workers in textile, dye, glass, petroleum, batteries, plastics, metal refining, barbershop and 64 others with limited exposure to HMTE. Smoking (ex/current) was noted in 72 patients. Exclusion criteria included history of previous neoplasm, chemo- or radiation therapy.

The controls included 51 age and sex matched normal individuals, from whom plasma and urine samples for HMTE and serum samples for OS and p38-MAPK marker assessment were taken. In addition, control bladder tissue samples were taken from 21 patients without BC, and used for comparison, regarding HMTE and MD gene. The latter patients included those subjected to ureteroneocystostomy or bladder augmentation.

3.1

Collection, processing, and digestion of HMTE plasma and urine samples

A 5-mL venous blood sample was taken in a pro-coagulation tube, 1 day before RC from BC patients, and the controls, kept at room temperature for 15 minutes, centrifuged at 4000 rpm for 10 minutes, and the plasma part was separated. A 20-mL of the patient urine in the early morning on the day of RC, and similar samples from the controls were collected in aliquot tubes. A mixture of 1-mL plasma or 2-mL urine and 3-mL of nitric acid (HNO3) and 1 ml of hydrogen peroxide (H2O2) is made and kept at room temperature for 15 minutes. The digestion was conducted by a microwave (Speed wave four, Berghof Products, Germany). After cooling, de-iodinised water was added to the digested sample up to 10-mL level.

3.2

Digestion of HMTE tissue samples

In BC patients and controls, the tissue samples were washed with distilled water, dried for 24 hours in a vacuum oven at 108°C, and then weighed and digested using a special microwave. Thereafter, an amount of hydrochloric and nitric acid (8 and 2 ml, respectively) was added to about a quarter of a gram of each sample, followed by heating up the mixture to 180°C with pressure for 30 min. In a 50-ml volumetric flask, the resulting solution was put and filled up to the mark using de-ionized water [ ].

3.3

HMTE measurement

The HMTE studied include Cd, Pb, Co, Ni, Sr, Al, Zn and B. HMTE were measured by the inductively coupled plasma optic emission spectrometry (iCAP™ 7400 ICP-OES Analyzer, ThermoFisher Scientific, Bremen, Germany). The tissue and plasma/urine concentrations of HMTE were usually reported in mg per kg and in microgram per liter (Ug/L), respectively and their limit of detection (LOD) were reported before [ , ]. Levels below LOD were stated as zero.

3.4

MD genes evaluation

The investigated MD genes included (mt-CO3, mt-CYB, mt-ATP 6, mt-ATP8, mt-CO1, mt-ND1). The genes were assessed by real-time quantitative polymerase chain reaction (RT-PCR). They were measured 3 times and an average was taken.

3.5

Total RNA extraction (mRNA)

RNA extraction from bladder tissue (cancerous, adjacent noncancerous and controls) was performed using miRNeasy Mini Kit (Cat. No. 217004, Qiagen, Hilden, Germany).

3.6

Convertion of RNA to cDNA

Thermal cycler PCR System (GeneAmp®PCR System 9700, Applied Biosystem, USA) was used for conversion of total RNA into complementary DNA (cDNA) with miRCURY LNA miRNA PCR Starter Kit (Cat. No. 339320, Qiagen) and QuantiNova Reverse Transcription Kit (Cat. No. 205411, Qiagen), according to manufacturer guidelines.

3.7

Quantitative RT-PCR for mRNA

The mRNAs of the investigated genes and the housekeeping gene GADPH (internal control) were evaluated by QuantiNova® SYBR® Green PCR KIT (Cat. No. 208052, Qiagen), QuantiNova LNA Primer Assays and Step one plus RT-PCR (Applied Biosystems). The equation ( RQ = 2 ^-ΔΔCT ) of the Leviak {the 2(-Delta Delta C(T)} method was used to analyze the relative gene quantification (RQ) [ ].

3.8

Evaluation of OS and MAPK markers

Enzyme-linked immunosorbent assay (ELISA) was used to measure these markers in the serum of patients and controls, where 3 measurements were conducted and an average was made. The markers included 8-OHdG, a marker of oxidative DNA damage, MDA, a marker of peroxidation (OS) of polyunsaturated fatty acids, 3-NT, a marker of protein oxidative/nitrative damage, AGEs, a marker of damage cell lipids and proteins, AOPP, a marker of protein oxidation, ROS, a major OS biomarker, SOD2, a mitochondrial antioxidant marker and p38-MAPK.

3.9

Statistical analysis

The software SPSS statistical package, version 21 (IBM SPSS Statistics) was used. Kolmogorov-Smirnov test was used to check the numerical data for normal versus non-normal distribution. Non-normally distributed variables were shown as median (interquartile range-IQR). Mann-Whitney and Kruskall-Wallis tests were used for comparison between groups with noncontinuous numerical data. Multiple tests, including Pearson’s, Kendall’s and Spearman’s test, were used for correlation among tissue, plasma and urine HMTE, in order to avoid type I error. ROC curve was plotted when indicated.