In Vitro and In Vivo Micronucleus Tests in Genotoxicological Research

U današnje vrijeme životni i radni okoliš čovjeka nepredvidiva je mješavina kemijskih spojeva od kojih neki mogu djelovati kao mutageni ili kao aneugeni karcinogeni. Osim toga, razni su izvori zračenja dodatno opterećenje na genom čovjeka. Od desetak metoda koje se rabe u genotoksikološkim istraživanjima, in vitro i in vivo mikronukleus tehnike posljednjih su godina našle primjenu kako u bazičnim tako i u kliničkim i farmakološkim istraživanjima. Prednost in vitro i in vivo mikronukleus tehnika je da istodobno pružaju podatke o klastogenom i aneugenom djelovanju agenasa. Kako ne zahtijeva kultiviranje stanica, in vivo mikronukleus tehnika, iako uvedena nekoliko godina nakon in vitro tehnike, zbog svoje jednostavnosti i brzine primjene postaje sve važniji izvor podataka u akutnim i kroničnim genotoksikološkim istraživanjima.Working and living environment today is an unpredictable mixture of different chemical substances. Some of these chemicals are mutagenic carcinogens and others are aneugenic carcinogens. An additional burden to the human genome is the exposure to several kinds of radiations. Among a dozen genotoxicological methods in use in vitro and in vivo, the micronucleus assay has found application in the basic research, clinical reasearch, and pharmacological studies. The advantage of in vivo and in vitro micronucleus assays is in the fact that they provide data on both clastogenic and aneugenic action of agents. Although several years behind the in vitro technique, the in vivo micronucleus assay has swiftly developed into a significant source of data in acute and chronic genotoxic investigations, as it does not require cell culture

Environmental and occupational exposures associated with male infertility

Porast slučajeva muške neplodnosti tijekom posljednja dva desetljeća, vjerojatno i zbog izloženosti okolišnim čimbenicima, izazvao je značajan interes. Izvješća klinika za neplodnost pokazala su da kronične bolesti i nasljedni čimbenici ili neke zdravstvene smetnje mogu samo djelomično objasniti trenutačnu incidenciju muške neplodnosti. Životni okoliš i radna okolina mogu imati značajnu ulogu u izlaganju složenim smjesama spojeva s endokrinim djelovanjem (ED), koji su povezani s raznim poremećajima plodnosti. Cilj je ovoga preglednog rada dati uvid u trenutačno znanje o tipovima izloženosti koji mogu biti povezani s muškom neplodnošću. Naše se istraživanje oslanjalo na sustavnu pretragu PubMeda, Scopusa i Web of Sciencea za članke objavljene od siječnja 2000. do rujna 2020. Rezultati su pokazali da je za neke čimbenike dobro dokumentirana povezanost s muškom neplodnošću, što uključuje pušenje, neke fiziološke poremećaje te kronične bolesti poput pretilosti i dijabetesa, koji sami po sebi mogu odražavati životne navike i izloženost okolišu, posebice spojevima s ED-om poput ftalata, bisfenola, pesticida i protupožarnih zaštita. Međutim, broj studija o etiologiji muške neplodnosti još je uvijek premalen u usporedbi s veličinom zahvaćene populacije. Medicina rada tijekom sistematskih pregleda ne prikuplja podatke o muškoj neplodnosti premda su spojevi s ED-om prisutni u mnogim tehnološkim procesima.The upsurge in male infertility over the last two decades, possibly due to environmental exposure, has raised significant interest, particularly boosted by reports from fertility clinics, which showed that chronic diseases and hereditary or other medical conditions might only partially explain current incidence of male infertility. Both environmental and occupational settings may have a significant role in exposure to complex mixtures of endocrine disruptors (ED), which play a major role in fertility disorders. The aim of this review is to give an insight into the current knowledge on exposure settings which may be associated with male infertility. Our study relied on a systematic search of PubMed, Scopus, and Web of Science for articles published between January 2000 and September 2020. It showed that some well documented factors associated with male infertility include smoking, and physiological disturbances or chronic diseases such as obesity and diabetes, which in turn, may also reflect lifestyle choices and environmental exposures, especially to EDs such as phthalates, bisphenols, pesticides, and flame retardants. However, the number of studies on the aetiology of male infertility is still too low in comparison with the size of affected population. Occupational health follow-ups and medical surveillance do not collect any data on male infertility, even though ED chemicals are part of many technological processes

Lipid peroxidation, detoxification capacity, and genome damage in mice after transplacental exposure to pharmaceutical drugs

Data on genome damage, lipid peroxidation, and levels of glutathione peroxidase (GPX) in newborns after transplacental exposure to xenobiotics are rare and insufficient for risk assessment. The aim of the current study was to analyze, in an animal model, transplacental genotoxicity, lipid peroxidation, and detoxification disturbances caused by the following drugs commonly prescribed to pregnant women: paracetamol, fluconazole, 5- nitrofurantoin, and sodium valproate. Genome damage in dams and their newborn pups transplacentally exposed to these drugs was investigated using the in vivo micronucleus (MN) assay. The drugs were administered to dams intraperitoneally in three consecutive daily doses between days 12 and 14 of pregnancy. The results were correlated, with detoxification capacity of the newborn pups measured by the levels of GPX in blood and lipid peroxidation in liver measured by malondialdehyde (HPLC-MDA) levels. Sodium valproate and 5-nitrofurantoin significantly increased MN frequency in pregnant dams. A significant increase in the MN frequency of newborn pups was detected for all drugs tested. This paper also provides reference levels of MDA in newborn pups, according to which all drugs tested significantly lowered MDA levels of newborn pups, while blood GPX activity dropped significantly only after exposure to paracetamol. The GPX reduction reflected systemic oxidative stress, which is known to occur with paracetamol treatment. The reduction of MDA in the liver is suggested to be an unspecific metabolic reaction to the drugs that express cytotoxic, in particular hepatotoxic, effects associated with oxidative stress and lipid peroxidation

Dobne i spolne razlike u oštećenju genoma između pretpubertetskih i odraslih miševa nakon izlaganja ionizirajućemu zračenju

The mechanisms that lead to sex and age differences in biological responses to exposure to ionising radiation and related health risks have still not been investigated to a satisfactory extent. The significance of sex hormones in the aetiology of radiogenic cancer types requires a better understanding of the mechanisms involved, especially during organism development. The aim of this study was to show age and sex differences in genome damage between prepubertal and adult mice after single exposure to gamma radiation. Genome damage was measured 24 h, 48 h, and 72 h after exposure of 3-week and 12-week old BALB/CJ mice to 8 Gy of gamma radiation using an in vivo micronucleus assay. There was a significantly higher genome damage in prepubertal than in adult animals of both sexes for all sampling times. Irradiation caused a higher frequency of micronuclei in males of both age groups. Our study confirms sex differences in the susceptibility to effects of ionising radiation in mice and is the first to show that such a difference occurs already at prepubertal age.Mehanizmi koji uzrokuju spolne i dobne razlike u biološkim odgovorima na izloženost ionizirajućemu zračenju i s tim u vezi zdravstvene rizike još nisu dovoljno ispitani. Kako bi se spoznao značaj spolnih hormona u etiologiji zračenjem izazvanih vrsta tumora, potrebno je bolje poznavanje mehanizama koji su uključeni u taj proces, osobito tijekom razvojne faze organizma. Cilj ovoga istraživanja bio je prikazati dobne i spolne razlike u oštećenju genoma između pretpubertetskih i odraslih miševa nakon jednokratnoga izlaganja gama-zračenju. Primjenom in vivo mikronukleus-testa izmjereno je oštećenje genoma nastalo 24 sata, 48 sati i 72 sata nakon izlaganja BALB/CJ miševa, starih tri tjedna i dvanaest tjedana, dozi gama zračenja od 8 Gy. U svim vremenskim točkama mjerenja uočeno je značajnije veće oštećenje genoma u pretpubertetskih u odnosu na odrasle jedinke obaju spolova. Zračenje je uzrokovalo veću učestalost mikronukleusa u muških jedinki u objema dobnim skupinama. Dobiveni rezultati potvrđuju postojanje spolnih razlika u osjetljivosti na učinke ionizirajućega zračenja u miševa, a ovo je prvo istraživanje rezultati kojega pokazuju da do takvih razlika dolazi već u pretpubertetskoj dobi

Prospektivno praćenje trudnica na monoterapiji lamotriginom u Hrvatskoj - predkoncepcijsko savjetovanje i praćenje lijekova

We prospectively surveyed 23 pregnant women with epilepsy on lamotrigine monotherapy and reported outcome of their pregnancies, including one fetal intrauterine death, one spontaneous abortion and two preterm deliveries. There were no congenital malformations in their offspring. Women with pregnancy planning and folic acid intake delivered babies with higher values of birth weight and birth length. There was large inter-patient variation during drug monitoring and in the need of dose adjustment. Individual approach to every woman and monotherapy with minimal effective lamotrigine dose with frequent drug monitoring enhances the possibility for successful pregnancy. The management of women with epilepsy should begin with pre-pregnancy counseling. Planned pregnancy enables periconceptional folic acid supplementation. Despite the small number of cases, these data indicate that lamotrigine treatment during pregnancy might be relatively safe. Larger prospective studies are needed to obtain adequate power for statistical analysis including long-term cohort studies.U ovoj studiji smo prospektivno pratili ishod trudnoće u 23 trudnice s epilepsijom koje su uzimale lamotrigin kao monoterapiju. Trudnoća je kod bolesnica rezultirala intrauterinom smrću djeteta u jednom slučaju. spontanim abortusom u jednom slučaju, te prijevremenim porodom u dva slučaja. Kod novorođenčadi nisu zabilježene kongenitalne malformacije. Žene koje su planirale trudnoću i uzimale folnu kiselinu rodile su djecu s višom tjelesnom masom i visinom. Postojala je velika različitost medu bolesnicama u praćenju doze lijeka te u potrebi za usklađivanjem doze. Veća je mogućnost uspješnog planiranja trudnoće ako se svakoj bolesnici pristupi individualno uz minimalnu djelotvornu dozu lijeka (lamotrigin). Liječenje trudnica treba započeti savjetovanjem prije začeća kada je moguće i pravodobno započeti s uzimanjem folne kiseline. Unatoč malom broju slučajeva podaci iz naše studije pokazuju kako liječenje lamotriginom tijekom trudnoće može biti relativno sigurno. Potrebne su veće prospektivne studije kako bi se postigla zadovoljavajuća statistička snaga dobivenih podataka

Hrvatski model integrativne prospektivne skrbi trudnoća u žena s epilepsijom

Epilepsy is the most common neurological complication in pregnancy. Women with epilepsy have a higher risk of complications in pregnancy. In Croatia, women with epilepsy are treated by neurologists at tertiary centers according to the place of residence. We prospectively followed-up pregnancies in women with epilepsy and healthy controls, and analyzed the factors responsible for their delivery outcomes and development of their babies. Healthy pregnant women had a higher level of education and economic status, but pregnant women with epilepsy took folic acid in a higher proportion than controls, possibly due to timely preconception counseling. Complications during pregnancy depended on the number of antiepileptic drugs and epilepsy control. We noticed some behavioral and cognitive aspects in children exposed in utero to valproic acid, which required follow up. The rate of congenital malformations was not increased. In conclusion, women with epilepsy should receive preconception counseling about the risk for pregnancy, but also about the possibilities to minimize that risk. We have introduced a model of integrative management of pregnancy and epilepsy based on close collaboration among different clinical experts in Croatia, in order to provide prompt counseling and timely intervention.Epilepsija je najčešća neurološka komplikacija u trudnoći. Žene s epilepsijom imaju veći rizik za komplikacije u trudnoći. U Hrvatskoj žene s epilepsijom obično prate neurolozi u tercijarnim centrima prema mjestu njihova boravka. Mi smo prospektivno pratili trudnoće u žena s epilepsijom i u zdravih kontrolnih trudnica te analizirali čimbenike odgovorne za ishode trudnoća i razvoj njihove djece. Zdrave trudnice su imale višu razinu obrazovanja i ekonomskog statusa, ali su žene s epilepsijom uzimale folnu kiselinu u višem postotku nego zdrave trudnice zahvaljujući pravodobnom predkoncepcijskom savjetovanju. Komplikacije za vrijeme trudnoće ovisile su o broju antiepileptičnih lijekova i kontroli epilepsije. Zamijetili smo određene bihevijoralne i kognitivne aspekte u djece izložene in utero valproičnoj kiselini, što zahtijeva daljnje praćenje. Stopa kongenitalnih malformacija nije bila povišena. U zaključku, ženama s epilepsijom trebali bismo omogućiti predkoncepcijsko savjetovanje o rizicima u trudnoći, ali i o mogućnostima kako značajno smanjiti taj rizik. Predstavili smo model integrativne prospektivne skrbi trudnica s epilepsijom koji se temelji na bliskoj suradnji različitih kliničkih eksperata u Hrvatskoj, u cilju osiguranja promptnog savjetovanja i pravodobne intervencije

Genotoxicity biomarkers in workers exposed to hexavalent chromium: a study under the European Human Biomonitoring Initiative

No âmbito da Iniciativa Europeia em Biomonitorização Humana (HBM4EU) realizou-se um estudo ocupacional, envolvendo trabalhadores com potencial exposição a crómio hexavalente [Cr( VI)], um reconhecido agente carcinogénico. No presente estudo são apresentados os resultados de biomarcadores de genotoxicidade, incluindo a análise de lesão no DNA e de alterações cromossómicas em células sanguíneas. O estudo foi realizado em vários Países Europeus e abrangeu trabalhadores de diversos setores industriais e atividades, bem como um grupo de controlo constituído por trabalhadores administrativos das mesmas empresas (controlo interno) e de outras não relacionadas com produção/aplicação de Cr( VI) (controlo externo). Os resultados mostraram níveis de alterações cromossómicas (ensaio do micronúcleo) e de lesão no DNA (ensaio do cometa) significativamente aumentados nos trabalhadores expostos comparativamente aos controlos externos ( p=0,03; p<0,001, respetivamente). Estes resultados sugerem que mesmo um baixo nível de exposição ao Cr( VI) representa um risco acrescido para a saúde dos trabalhadores e, principalmente, para os que realizam cromagem em banho. O grupo controlo interno apresentou níveis médios de lesões no DNA e nos cromossomas comparáveis aos do grupo exposto, salientando a relevância de se considerarem também em risco. O uso de biomarcadores de efeito demonstrou ser crucial para a deteção precoce de efeitos biológicos decorrentes de baixos níveis de exposição ao Cr( VI), contribuindo para a identificação de subgrupos em maior risco. O presente estudo vem apoiar a necessidade de uma reavaliação do limite de exposição ocupacional a Cr( VI), bem como da implementação de medidas de gestão de risco conducentes a uma melhor proteção da saúde dos trabalhadores.An occupational study was carried out within the scope of the European Initiative on Human Biomonitoring (HBM4EU), involving workers with potential exposure to hexavalent chromium [Cr(VI)], a recognized carcinogenic agent. In the present study, the results of biomarkers of genotoxicity are presented, including the analysis of DNA damage and chromosomal alterations in blood cells. The study was carried out in several countries and included workers from different industrial sectors and activities, as well as a control group including administrative workers from the same companies (internal control) and from others not related to the production/application of Cr(VI) (external control). Results showed significantly increased levels of chromosomal alterations (micronucleus assay) and DNA damage (comet assay) in exposed workers compared to external controls (p=0.03; p<0.001, respectively). These results suggest that even a low level of exposure to Cr(VI) represents an increased risk for the health of workers and, mainly, for those who per form chromium bath plating. The internal control group displayed DNA and chromosomal damage levels comparable to those of the exposed group, highlighting the impor tance of also being considered as potentially at risk. The use of ef fect biomarkers proved to be crucial for the early detection of biological ef fects resulting from low levels of exposure to Cr(VI), contributing to the identification of subgroups at a higher risk. The present study suppor ts the need for a reassessment of the occupational exposure limit to Cr(VI), as well as the implementation of risk management measures leading to a better protection of workers' health.Este projeto recebeu financiamento do programa de investigação e inovação Horizonte 2020 da União Europeia sob o acordo de subvenção n.º 733032 e recebeu cofinanciamento das organizações dos autores.info:eu-repo/semantics/publishedVersio