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The isolation of keratin from poultry feathers using subcritical water was studied in a batch reactor at temperatures (120–250 ◦C) and reaction times (5–75 min). The hydrolyzed product was characterized by FTIR and elemental analysis, while the molecular weight of the isolated product was determined by SDS-PAGE electrophoresis. To determine whether disulfide bond cleavage was followed by depolymerization of protein molecules to amino acids, the concentration of 27 amino acids in the hydrolysate was analyzed by GC/MS. The optimal operating parameters for obtaining a high molecular weight protein hydrolysate from poultry feathers were 180 ◦C and 60 min. The molecular weight of the protein hydrolysate obtained under optimal conditions ranged from 4.5 to 12 kDa, and the content of amino acids in the dried product was low (2.53% w/w). Elemental and FTIR analyses of unprocessed feathers and dried hydrolysate obtained under optimal conditions showed no significant differences in protein content and structure. Obtained hydrolysate is a colloidal solution with a tendency for particle agglomeration. Finally, a positive influence on skin fibroblast viability was observed for the hydrolysate obtained under optimal processing conditions for concentrations below 6.25 mg/mL, which makes the product interesting for various biomedical applications.

Prostate cancer is among the most commonly diagnosed malignancies in men and its incidence increases with age. In addition to family history, race, and ethnicity, various external risk factors may contribute to its increased risk. Arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) are pervasive environmental contaminants and several experimental and epidemiological studies linked exposure to these metal(loid)s with an increased risk of prostate cancer. However, research on the impact of low-level exposure is scanty and largely inconclusive. Prostate specific antigen (PSA), combined with a digital rectal examination of prostate, has been routinely used for early detection of prostate cancer in spite of its insufficient specificity for clinically important cancers. Moreover, several studies in presumably healthy men indicated that various external factors can affect PSA levels, including metal(loid) exposure. Therefore, we aimed to evaluate the role of environmental exposure to As, Cd, Pb and Hg in prostate cancer risk and their impact on serum PSA. Blood As, Cd, Pb, and Hg and serum PSA were measured in 62 prostate cancer patients and 30 control men with no occupational exposure to metals. Prostate cancer patients had significantly higher serum PSA and blood Hg than control subjects. The results of Spearman’s correlation and a simple linear regression analysis in all 92 subjects showed a positive association between blood Hg and PSA. However, after control for the impact of the remaining elements in the models of multiple regression, this association was lost. Although we did not confirm a linear association between measured metal(loid)s and PSA in conditions of environmental exposure, further investigation in larger cohorts, taking into consideration possible synergistic or non-linear mixture effects, are warranted.

Testicular germ cell tumours (TGCT) are the most common malignancy in men aged 15–35 years and its incidence is increasing worldwide. Tumour markers commonly used in the diagnosis and prognosis of TGCT have limited sensitivity and specificity and additional, minimally invasive biomarkers for disease management are needed. Metabolomic-based profiling of amino acids in urine may identify changed metabolites as potential biomarkers of testicular cancer. Impaired metal(loid) homeostasis may increase susceptibility to cancer development. We compared a quantitative profile of 30 amino acids and 18 metal(loid)s in urine samples between 86 men with newly diagnosed TGCT and 68 control subjects, aged 18 to 55 years. The concentration of free amino acids was determined by GC-MS using Phenomenex EZ:faast kit for sample preparation, multi-element analysis was performed by ICP-MS, and all results were corrected for creatinine levels. Testicular cancer patients had significantly higher levels of aspartic acid (Asp), manganese (Mn) and zinc (Zn), and significantly lower levels of threonine (Thr), serine (Ser), histidine (His) and cobalt (Co) when compared to control subjects. Linear discriminant analysis using all 30 amino acids successfully separated subjects with TGCT from control subjects and the discrimination rate was 86%. Metabolic pathway analysis (MetaboAnalyst 4.0) showed that Asp, Thr, Ser, and His were related to alterations in 12 metabolic pathways and those with the highest impact were metabolism of alanine, aspartate and glutamate (0.26) and glycine, Thr and Ser (0.23). Our results indicated that disorders in the citric acid cycle, the urea cycle and amino acid metabolism play a role in the pathogenesis of testicular cancer. The impaired urinary profile of Mn, Co and Zn may be highly relevant with regard to their role in amino acids synthesis, antioxidant defense and genetic repair processes. More research is needed to verify our findings

Maternal cigarette smoking may impair the pro/antioxidant balance, affect bodily element disposition and disturb nutrient and oxygen transport to the developing foetus. The aim of this study was to assess the effect of active maternal smoking during pregnancy on superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and metallothionein (MT), cadmium (Cd), and zinc (Zn) concentrations in the samples of maternal and cord blood and placenta of healthy mother-newborn pairs collected after term vaginal delivery. We compared results for non-smokers and smokers (n = 37 each), grouped based on cotinine urine levels as a biomarker of tobacco smoke exposure (100 ng/mL in active smokers). Activity of SOD was 14 % lower in maternal plasma, 91 % higher in placenta and 12 % lower in cord plasma of smokers compared to non-smokers, whereas GPx and MT did not differ between the groups. These results accompanied decreased maternal blood Zn and increased placental Zn in smokers who also had significantly higher Cd in maternal blood and placenta than non-smokers. Cord blood Cd was low and did not differ between the groups. Although active maternal smoking contributed to Cd levels in maternal blood and placenta and Zn in placenta, there was no induction of increased placental MT synthesis. Overexpression of placental SOD can increase the susceptibility of offspring to oxidative stress and risk of disease development, even later in life. The study was funded by the Croatian Science Foundation (grant HRZZ-IP-2016-06-1998).

Tumor testisa najčešće je maligno oboljenje u muškaraca mlađe životne dobi. Razvojem tumora nastaju promjene u metabolizmu stanica koje se mogu pratiti analizom metabolita u biološkim uzorcima ispitanika što može biti koristan alat za procjenu stanja organizma i daljnje liječenje bolesti. Cilj ovog istraživanja bio je kreirati kvantitativni profil slobodnih aminokiselina i elemenata u urinu ispitanika s tumorom testisa i odrediti vrijednosti parametara oksidacijsko-redukcijske ravnoteže u krvi. U ispitanika s tumorom testisa izmjerene su značajno povišene koncentracije asparaginske kiseline, mangana i cinka te aktivnost superoksid dismutaze dok su koncentracija treonina, serina, histidina, kobalta i glutationa te ukupni oksidacijski status, bili značajno niži u odnosu na kontrolne ispitanike. Nadalje, provedena je linearna diskriminantna analiza kako bi se ispitalo razdvaja li kreirani profil aminokiselina ispitanike s tumorom testisa od kontrolnih ispitanika, a uspješnost diskriminacije iznosila je 86 %. U urinu ispitanika s tumorom testisa kreiran je i kvalitativni profil hlapljivih organskih metabolita kako bi detektirali koje se vrste, ovisno o funkcionalnim skupinama, javljaju u urinu. Analizom urina u kromatogramu je detektirano oko 200 pikova hlapljivih organskih metabolita te su identificirana 123 metabolita. Procijenjen je učinak kemoterapije cisplatinom na koncentraciju aminokiselina i elemenata te na parametre oksidacijsko-redukcijske ravnoteže. Nakon primljene kemoterapije, u ispitanika s tumorom testisa izmjerene su povišene koncentracije većine aminokiselina i elemenata u urinu te glutationa u krvnoj plazmi u odnosu na vrijednosti prije kemoterapije. Godinu dana nakon kemoterapije rezultati su pokazali slične vrijednosti za većinu aminokiselina osim za treonin, serin i živu koji su bili značajno sniženi, te mangan i platinu koji su i dalje bili značajno povišeni kod ispitanika koji su bolovali od tumora testisa u odnosu na kontrolne ispitanike. Rezultati upućuju na postupni oporavak metabolizma nakon prestanka kemoterapije. Ovim istraživanjem dobiveni su rezultati koji upućuju na to da razvojem tumora u testisima dolazi do poremećaja u ciklusu limunske kiseline i ciklusu uree. Dobiveni rezultati mogu poslužiti kao temelj za daljnja istraživanja o ulozi međuprodukata navedenih ciklusa u tumorigenezi što može pridonijeti eventualnom otkrivanju novih bioloških biljega tumora testisa. Testicular cancer is the most common malignancy in young men and in recent decades its occurrence has been growing rapidly. Tumour development induces changes in cell metabolism that can be monitored by analysing the metabolites in biological samples of the subject. The aim of this study was to create a quantitative profile of free amino acids and elements in urine and levels of oxidative stress parameters in blood of men with testicular cancer and compare them with control subjects. In subjects with testicular cancer, significantly higher concentrations of aspartic acid, manganese and zinc were measured, as well as a higher activity of superoxide dismutase. On the other hand, concentrations of threonine, serine, histidine, cobalt and glutathione and total oxidant status were significantly lower in comparison to control subjects. Additionally, linear discriminant analysis (LDA) was performed to examine whether the created amino acid profile discriminated subjects with testicular cancer from control subjects and the results showed that the discrimination rate was 86%. In the urine of subjects with testicular cancer a qualitative profile of volatile organic metabolites was also created to detect which species, depending on their functional groups, can be detected in urine. GCMS analysis of urine detected about 200 peaks of volatile organic metabolites, and 123 of them were identified. The effect of cisplatin chemotherapy on the concentration of amino acids and elements in urine and on the parameters of oxidative stress was also estimated. After receiving chemotherapy, concentrations of the majority of amino acids and elements in urine and glutathione in blood plasma increased in comparison to values obtained before chemotherapy. One year after receiving chemotherapy the results showed that the concentrations of most amino acids and elements were similar to those measured in the control subjects, except for threonine, serine and mercury, which had significantly higher values, and manganese and platinum, which had significantly lower values than the control subjects. Results indicated a gradual metabolism recover following chemotherapy. The results of this research suggest that the development of a tumour in the testicles leads to a disorder in the citric acid cycle and the urea cycle. The obtained results can be used as a basis for further research on the role of the intermediates of the above-mentioned cycles in tumorigenesis, which could contribute to the possible discovery of new biomarkers for testicular tumours.

Dosadašnja epidemiološka istraživanja raka testisa pokazala su da je u uvjetima profesionalne izloženosti elementima poput As, Cd, Ni i Pb značajno povećan rizik za razvoj ove bolesti. Literaturni podaci ukazali su također na štetno djelovanje pojedinih elemenata u uvjetima niske do umjerene razine izloženosti, osobito kada je ta izloženost dugotrajna. S obzirom na to da su elementi široko rasprostranjeni u čovjekovu okolišu, čovjek im je neizbježno izložen. Stoga je važno razmatrati i utjecaj elemenata u uvjetima ekološke, relativno niske razine izloženosti, kao i utjecaj istovremene izloženosti različitim elementima. Cilj ovog istraživanja bio je izmjeriti koncentraciju Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Pt, Se, Sn i Zn u punoj krvi, serumu i urinu muškaraca s germinativnim tumorima testisa koji nisu profesionalno izloženi metalima i dobivene vrijednosti usporediti s onima izmjerenima u zdravih muškaraca. Profil elemenata u krvi, serumu i urinu i učinak koji oni mogu imati na razine tumorskih biljega do sada nisu proučavani u muškaraca s rakom testisa. Uspješnost diskriminacije ispitanika s rakom testisa u odnosu na kontrolne ispitanike temeljem profila svih mjerenih elemenata iznosila je preko 95% za sve tri biološke matrice (krv, serum i urin). Rezultati linearne diskriminantne analize (LDA) izdvojili su izmjerene koncentracije Ca, Co, Cu, Fe, K, Mg, Na i Zn u krvi, Ca, Cu, Na i Ni u serumu, te koncentraciju Cd, Co, Fe i Mn u urinu kao najjače prediktore bolesti. Utvrđena je značajna pozitivna korelacija između koncentracije α-fetoproteina (AFP) i Cu, β- humanog korionskog gonadotropina (β-HCG) i Cu te povezanost laktat dehidrogenaze (LDH) s koncentracijom Cu, Ca i K u serumu ispitanika s rakom testisa. Utvrđena je statistički značajna inverzna povezanost između koncentracije Fe u serumu s razinama AFP i LDH, kao i značajna inverzna povezanost između LDH i Se. S obzirom na važnu ulogu ispitivanih elemenata u mehanizmima koji određuju konačan ishod štetnog djelovanja različitih agenasa na ljudsko zdravlje, poremećaj homeostaze toksičnih i esencijalnih elemenata mogao bi dovesti do povećane osjetljivosti organizma i doprinijeti razvoju raka testisa, čak i u uvjetima umjerene do niske razine izloženosti.

Nastanak tumorskih stanica povezan je s brojnim biokemijskim i molekularnim promjenama u organizmu čije uočavanje ima veliki potencijal u ranoj dijagnostici i liječenju. Tumori, kao abnormalne nakupine tkiva, oduzimaju organizmu hranjive tvari, kisik, hormone i ostale životno bitne sastojke u svrhu vlastitog rasta te tako utječu na metabolizam stanice i regulaciju njene funkcije. Poznavanje i razumijevanje metaboličkih razlika između normalnih stanica i stanica raka prepoznato je kao moguća meta za ubijanje tumorskih stanica. Cilj ovog istraživanja bio je usporediti kvantitativni profil slobodnih aminokiselina i elemenata u urinu ispitanika s tumorom testisa u odnosu na njihove vrijednosti u kontrolnih ispitanika. Koncentracije slobodnih aminokiselina izmjerene su primjenom GC-MS-a nakon priprave uzoraka korištenjem komercijalnog seta reagensa Phenomenex EZ:faast, a koncentracije elemenata izmjerene su primjenom ICP-MS-a. U ispitanika s tumorom testisa izmjerene su značajno povišene koncentracije asparaginske kiseline, mangana i cinka, a značajno snižene koncentracije treonina, serina, histidina i kobalta u odnosu na koncentracije izmjerene u kontrolnih ispitanika. Ovi rezultati upućuju na to da razvojem tumora u testisima dolazi do poremećaja u ciklusu limunske kiseline i ciklusu uree. Naime, asparaginska kiselina ima važnu ulogu u ciklusu limunske kiseline i ciklusu uree. Dosadašnja istraživanja su pokazala da asparaginska kiselina sudjeluje u proliferaciji, iako ne svih vrsta tumora. Kobalt je neophodna komponenta kobalamina (vitamina B12) i ima vrlo važnu ulogu u formiranju aminokiselina, dok kobalaminski enzimi kataliziraju reakcije nastanka međuprodukata koji ulaze u ciklus limunske kiseline. Mangan ima ključnu ulogu u mnogim staničnim procesima, uključujući metabolizam lipida, proteina i ugljikohidrata te je sastavni dio enzima arginaze uključenih u sintezu uree i metabolizam piruvata. Dobiveni rezultati mogu poslužiti kao temelj za daljnja istraživanja o ulozi međuprodukata navedenih ciklusa u tumorigenezi što može pridonijeti eventualnom otkrivanju novih bioloških biljega tumora testisa.

We investigated the effect of low-dose exposure to glyphosate on 3 months old male Wistar rats that received orally for 28-day doses of 0.1 mg kg-1 b.w. of the acceptable operator exposure level-(AOEL), 0.5 mg kg-1 b.w. consumers acceptable daily intake (ADI), 1.75 mg kg-1 b.w. of the chronic population-adjusted dose (cPAD), 10 mg kg-1 b.w. (100xAOEL) and the same volume of phosphate buffer without glyphosate (negative control). Positive control received 300 mg kg-1 b.w. of ethyl methanesulphonate (EMS) over the last three days of experiment.The samples of whole blood, plasma and liver samples were analyzed for the levels of plasma cholinesterase activity, oxidative stress (lipid peroxidation (thiobarbituric reactive substances, TBARS), glutathione(GSH), glutathione peroxidase (GSH-Px), reactive oxidative species (ROS)) and DNA damage in whole blood and small (non-parenchymal cells) and medium (parenchymal cells-hepatocytes) liver cells.The exposed animals gained less weight than control. The cPAD and the 100xAOEL groups' liver weight increased. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. Exposure lowered TBARS values in all exposed groups, with significant liver changes in the AOEL, ADI, and cPAD groups and plasma changes in the AOEL and cPAD group. Exposure also produced significant primary DNA damage in the leukocytes and liver cells. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition.This work was financially supported by Project Organic Pollutants in Environment – Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation (grant number 8366).

Prostate cancer is among the most common cancers diagnosed in men in developed countries. The imbalance between toxic and essential elements and mechanisms of antioxidant defence in the organism can lead to increased sensitivity and contribute to the development of this disease. Data concerning the levels of trace elements and antioxidant system parameters in men with prostate cancer are limited as their results are conflicting. In this study we determined the concentration of several toxic and/or essential trace elements and the activity of antioxidative enzymes copper, zinc-superoxide dismutase (Cu, Zn-SOD) and glutathione peroxidase (GSH-Px) in blood obtained from 53 patients with newly diagnosed prostate cancer and from 53 healthy volunteers. In subjects with newly diagnosed prostate cancer we found significantly higher concentration of lead, mercury and molybdenum in blood and serum, arsenic and chromium in serum and copper and iron in blood, as well as significantly higher activity of Cu, Zn-SOD and GSH-Px. Subjects with prostate cancer had significantly lower concentration of serum zinc, blood manganese, and cobalt in both serum and whole blood compared to healthy volunteers. Elevated levels of toxic elements (such as lead, mercury and arsenic) measured in patients with prostate cancer suggest that even low exposure to these elements may present a risk for the development of cancer, probably via formation of reactive oxygen species and/or reducing the bioavailability of zinc. The increased enzymatic activity is most likely the result of the cell response to oxidative mechanisms involved in the development and progression of prostate cancer.