Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours. © 2004 Cancer Research UK.

Hypothesis: is Alzheimer's disease a metal-induced immune disorder?

A hypothesis that a metal-induced immune disorder may be involved in the pathogenesis of some forms of Alzheimer's disease (AD) is presented. The classical complement pathway is activated in AD and T cells and reactive microglia appear in the brain. Studies of metal induced autoimmunity and the use of compounds containing aluminium as vaccine adjuvants suggest that metals can activate complement and can be taken up by antigen presenting cells. The consequent immune response could contribute to neuronal damage, beta-amyloid deposition and cell death. The strengths and weaknesses of this hypothesis are discussed and tests of some aspects are proposed.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

The predictive ability of clinical tests for contact lens induced dry eye

Approximately half of current contact lens wearers suffer from dryness and discomfort, particularly towards the end of the day. Contact lens practitioners have a number of dry eye tests available to help them to predict which of their patients may be at risk of contact lens drop out and advise them accordingly. This thesis set out to rationalize them to see if any are of more diagnostic significance than others. This doctorate has found: (1) The Keratograph, a device which permits an automated, examiner independent technique for measuring non invasive tear break up time (NITBUT) measured NITBUT consistently shorter than measurements recorded with the Tearscope. When measuring central corneal curvature the spherical equivalent power of the cornea was measured as being significantly flatter than with a validated automated keratometer. (2) Non-invasive and invasive tear break-up times significantly correlated to each other, but not the other tear metrics. Symptomology, assessed using the OSDI questionnaire, correlated more with those tests indicating possible damage to the ocular surface (including LWE, LIPCOF and conjunctival staining) than with tests of either tear volume or stability. Cluster analysis showed some statistically significant groups of patients with different sign and symptom profiles. The largest cluster demonstrated poor tear quality with both non-invasive and invasive tests, low tear volume and more symptoms. (3) Care should be taken in fitting patients new to contact lenses if they have a NITBUT less than 10s or an OSDI comfort rating greater than 4.2 as they are more likely to drop-out within the first 6 months. Cluster analysis was not found to be beneficial in predicting which patients will succeed with lenses and which will not. A combination of the OSDI questionnaire and a NITBUT measurement was most useful both in diagnosing dry eye and in predicting contact lens drop out.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

The discrimination and association of float glass and the quantitative analysis of liquids from aerosols and microdrops using laser induced breakdown spectroscopy

Glass is a common form of trace evidence found at many scenes of crimes in the form of small fragments. These glass fragments can transfer to surrounding objects and/or persons and may provide forensic investigators valuable information to link a suspect to the scene of a crime. Since the elemental composition of different glass sources can be very similar, a highly discriminating technique is required to distinguish between fragments that have originated from different sources. ^ The research presented here demonstrates that Laser Induced Breakdown Spectroscopy (LIBS) is a viable analytical technique for the association and discrimination of glass fragments. The first part of this research describes the optimization of the LIBS experiments including the use of different laser wavelengths to investigate laser-material interaction. The use of a 266 nm excitation laser provided the best analytical figures of merit with minimal damage to the sample. The resulting analytical figures of merit are presented. The second part of this research evaluated the sensitivity of LIBS to associate or discriminate float glass samples originating from the same manufacturing plants and produced at approximately the same time period. Two different sample sets were analyzed ranging in manufacturing dates from days to years apart. Eighteen (18) atomic emission lines corresponding to the elements Sr, K, Fe, Ca, Al, Ba, Na, Mg and Ti, were chosen because of their detection above the method detection limits and for presenting differences between the samples. Ten elemental ratios producing the most discrimination were selected for each set. When all the ratios are combined in a comparison, 99% of the possible pairs were discriminated using the optimized LIBS method generating typical analytical precisions of ∼5% RSD. ^ The final study consisted of the development of a new approach for the use of LIBS as a quantitative analysis of ultra-low volume solution analysis using aerosols and microdrops. Laser induced breakdown spectroscopy demonstrated to be an effective technique for the analysis of as low as 90 pL for microdrop LIBS with 1 pg absolute LOD and 20 µL for aerosol LIBS with an absolute LOD of ∼100 fg.^

Effects of architectural features of air-permeable roof cladding materials on wind-induced uplift loading

Widespread damage to roofing materials (such as tiles and shingles) for low-rise buildings, even for weaker hurricanes, has raised concerns regarding design load provisions and construction practices. Currently the building codes used for designing low-rise building roofs are mainly based on testing results from building models which generally do not simulate the architectural features of roofing materials that may significantly influence the wind-induced pressures. Full-scale experimentation was conducted under high winds to investigate the effects of architectural details of high profile roof tiles and asphalt shingles on net pressures that are often responsible for damage to these roofing materials. Effects on the vulnerability of roofing materials were also studied. Different roof models with bare, tiled, and shingled roof decks were tested. Pressures acting on both top and bottom surfaces of the roofing materials were measured to understand their effects on the net uplift loading. The area-averaged peak pressure coefficients obtained from bare, tiled, and shingled roof decks were compared. In addition, a set of wind tunnel tests on a tiled roof deck model were conducted to verify the effects of tiles' cavity internal pressure. Both the full-scale and the wind tunnel test results showed that underside pressure of a roof tile could either aggravate or alleviate wind uplift on the tile based on its orientation on the roof with respect to the wind angle of attack. For shingles, the underside pressure could aggravate wind uplift if the shingle is located near the center of the roof deck. Bare deck modeling to estimate design wind uplift on shingled decks may be acceptable for most locations but not for field locations; it could underestimate the uplift on shingles by 30-60%. In addition, some initial quantification of the effects of roofing materials on wind uplift was performed by studying the wind uplift load ratio for tiled versus bare deck and shingled versus bare deck. Vulnerability curves, with and without considering the effects of tiles' cavity internal pressure, showed significant differences. Aerodynamic load provisions for low-rise buildings' roofs and their vulnerability can thus be more accurately evaluated by considering the effects of the roofing materials.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Charge migration induced by attosecond pulses in bio-relevant molecules

After sudden ionization of a large molecule, the positive charge can migrate throughout the system on a sub-femtosecond time scale, purely guided by electronic coherences. The possibility to actively explore the role of the electron dynamics in the photo-chemistry of bio-relevant molecules is of fundamental interest for understanding, and perhaps ultimately controlling, the processes leading to damage, mutation and, more generally, to the alteration of the biological functions of the macromolecule. Attosecond laser sources can provide the extreme time resolution required to follow this ultrafast charge flow. In this review we will present recent advances in attosecond molecular science: after a brief description of the results obtained for small molecules, recent experimental and theoretical findings on charge migration in bio-relevant molecules will be discussed.

Influence of grinding operations on surface integrity and chloride induced stress corrosion cracking of stainless steels

Stainless steels were developed in the early 20th century and are used where both the mechanical properties of steels and corrosion resistance are required. There is continuous research to allow stainless steel components to be produced in a more economical way and be used in more harsh environments. A necessary component in this effort is to correlate the service performance with the production processes. The central theme of this thesis is the mechanical grinding process.  This is commonly used for producing stainless steel components, and results in varied surface properties that will strongly affect their service life. The influence of grinding parameters including abrasive grit size, machine power and grinding lubricant were studied for 304L austenitic stainless steel (Paper II) and 2304 duplex stainless steel (Paper I). Surface integrity was proved to vary significantly with different grinding parameters. Abrasive grit size was found to have the largest influence. Surface defects (deep grooves, smearing, adhesive/cold welding chips and indentations), a highly deformed surface layer up to a few microns in thickness and the generation of high level tensile residual stresses in the surface layer along the grinding direction were observed as the main types of damage when grinding stainless steels. A large degree of residual stress anisotropy is interpreted as being due to mechanical effects dominating over thermal effects. The effect of grinding on stress corrosion cracking behaviour of 304L austenitic stainless steel in a chloride environment was also investigated (Paper III). Depending on the surface conditions, the actual loading by four-point bend was found to deviate from the calculated value using the formula according to ASTM G39 by different amounts. Grinding-induced surface tensile residual stress was suggested as the main factor to cause micro-cracks initiation on the ground surfaces. Grinding along the loading direction was proved to increase the susceptibility to chloride-induced SCC, while grinding perpendicular to the loading direction improved SCC resistance. The knowledge obtained from this work can provide a reference for choosing appropriate grinding parameters when fabricating stainless steel components; and can also be used to help understanding the failure mechanism of ground stainless steel components during service.

The role of intracellular calcium perturbations in muscle damage and dysfunction in mouse models of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. DMD is clinically characterized by severe, progressive and irreversible loss of muscle function, in which most patients lose the ability to walk by their early teens and die by their early 20’s. Impaired intracellular calcium (Ca2+) regulation and activation of cell degradation pathways have been proposed as key contributors to DMD disease progression. This dissertation research consists of three studies investigating the role of intracellular Ca2+ in skeletal muscle dysfunction in different mouse models of DMD. Study one evaluated the role of Ca2+-activated enzymes (proteases) that activate protein degradation in excitation-contraction (E-C) coupling failure following repeated contractions in mdx and dystrophin-utrophin null (mdx/utr-/-) mice. Single muscle fibers from mdx/utr-/- mice had greater E-C coupling failure following repeated contractions compared to fibers from mdx mice. Moreover, protease inhibition during these contractions was sufficient to attenuate E-C coupling failure in muscle fibers from both mdx and mdx/utr-/- mice. Study two evaluated the effects of overexpressing the Ca2+ buffering protein sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) in skeletal muscles from mdx and mdx/utr-/- mice. Overall, SERCA1 overexpression decreased muscle damage and protected the muscle from contraction-induced injury in mdx and mdx/utr-/- mice. In study three, the cellular mechanisms underlying the beneficial effects of SERCA1 overexpression in mdx and mdx/utr-/- mice were investigated. SERCA1 overexpression attenuated calpain activation in mdx muscle only, while partially attenuating the degradation of the calpain target desmin in mdx/utr-/- mice. Additionally, SERCA1 overexpression decreased the SERCA-inhibitory protein sarcolipin in mdx muscle but did not alter levels of Ca2+ regulatory proteins (parvalbumin and calsequestrin) in either dystrophic model. Lastly, SERCA1 overexpression blunted the increase in endoplasmic reticulum stress markers Grp78/BiP in mdx mice and C/EBP homologous protein (CHOP) in mdx and mdx/utr-/- mice. Overall, findings from the studies presented in this dissertation provide new insight into the role of Ca2+ in muscle dysfunction and damage in different dystrophic mouse models. Further, these findings support the overall strategy for improving intracellular Ca2+ control for the development of novel therapies for DMD.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.