Novel In Vivo CometChip Reveals NDMA-Induced DNA Damage and Repair in Multiple Mouse Tissues.

The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent N-nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.

Hypometabolism and hypothermia in the rat model of endotoxic shock: independence of circulatory hypoxia.

We tested the hypothesis that development of hypothermia instead of fever in endotoxic shock is consequential to hypoxia. Endotoxic shock was induced by bacterial lipopolysaccharide (LPS, 500 µg kg(-1) i.v.) in rats at an ambient temperature of 22 °C. A ß3-adrenergic agonist known to activate metabolic heat production, CL316,243, was employed to evaluate whether thermogenic capacity could be impaired by the fall in oxygen delivery (DO2) during endotoxic shock. This possibility was rejected as CL316,243 (0.15 mg kg(-1) i.v.) evoked similar rises in oxygen consumption (V̇O2) in the presence and absence of endotoxic shock. Next, to investigate whether a less severe form of circulatory hypoxia could be triggering hypothermia, the circulating volume of LPS-injected rats was expanded using 6% hetastarch with the intention of improving tissue perfusion and alleviating hypoxia. This intervention attenuated not only the fall in arterial pressure induced by LPS, but also the associated falls in V̇O2 and body temperature. These effects, however, occurred independently of hypoxia, as they were not accompanied by any detectable changes in NAD(+)/NADH ratios. Further experimentation revealed that even the earliest drops in cardiac output and DO2 during endotoxic shock did not precede the reduction in V̇O2 that brings about hypothermia. In fact, DO2 and V̇O2 fell in such a synchrony that the DO2/V̇O2 ratio remained unaffected. Only when hypothermia was prevented by exposure to a warm environment (30 °C) did an imbalance in the DO2/V̇O2 ratio become evident, and such an imbalance was associated with reductions in the renal and hypothalamic NAD(+)/NADH ratios. In conclusion, hypometabolism and hypothermia in endotoxic shock are not consequential to hypoxia but serve as a pre-emptive strategy to avoid hypoxia in this model.

Lipopolysaccharide-induced hypothermia and hypotension are associated with inflammatory signaling that is triggered outside the brain.

Little is known about the neuroimmune mechanisms responsible for the switch from fever to hypothermia observed in severe forms of systemic inflammation. We evaluated whether bacterial lipopolysaccharide (LPS) acting directly on the brain could promote a fever-hypothermia switch as well as the hypotension that is often associated with hypothermia in models of systemic inflammation. At an ambient temperature of 22°C, freely moving rats received intracerebroventricular (i.c.v.) injections of LPS at doses ranging from 0.5 to 25µg. Despite the use of such high doses, the prevailing thermal response was fever. To investigate if a hypothermic response could be hidden within the prevailing febrile response, rats were pretreated with a cyclooxygenase-2 inhibitor (SC-236, 3.5mg/kg i.v.) known to block fever, but this strategy also failed to reveal any consistent hypothermic response following i.c.v. LPS. At the doses tested, i.c.v. LPS was similarly ineffective at inducing hypotension. Additional doses of LPS did not need to be tested because the 25-µg dose was already sufficient to induce both hypothermia and hypotension when administered peripherally (intra-arterially). An empirical 3D model of the interplay among body temperature, arterial pressure and heart rate following intra-arterial LPS reinforced the strong association of hypothermia with hypotension and, at the same time, exposed a bell-shaped relationship between heart rate and body temperature. In summary, the present study demonstrates that hypothermia and hypotension are triggered exclusively by LPS acting outside the brain and provides an integrated model of the thermal and cardiovascular responses to peripheral LPS.

Molecular origins of mutational spectra produced by the environmental carcinogen N-nitrosodimethylamine and SN1 chemotherapeutic agents.

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O 6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation.

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.

Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice.

N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag-/-) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage.

Inflammation, necrosis, and the kinase RIP3 are key mediators of AAG-dependent alkylation-induced retinal degeneration.

DNA-alkylating agents are commonly used to kill cancer cells, but the base excision repair (BER) pathway they trigger can also produce toxic intermediates that cause tissue damage, such as retinal degeneration (RD). Apoptosis, a process of programmed cell death, is assumed to be the main mechanism of this alkylation-induced photoreceptor (PR) cell death in RD. Here, we studied the involvement of necroptosis (another programmed cell death process) and inflammation in alkylation-induced RD. Male mice exposed to a methylating agent exhibited a reduced number of PR cell rows, active gliosis, and cytokine induction and macrophage infiltration in the retina. Dying PRs exhibited a necrotic morphology, increased 8-hydroxyguanosine abundance (an oxidative damage marker), and overexpression of the necroptosis-associated genes Rip1 and Rip3 The activity of PARP1, which mediates BER, cell death, and inflammation, was increased in PR cells and associated with the release of proinflammatory chemokine HMGB1 from PR nuclei. Mice lacking the anti-inflammatory cytokine IL-10 exhibited more severe RD, whereas deficiency of RIP3 (also known as RIPK3) conferred partial protection. Female mice were partially protected from alkylation-induced RD, showing reduced necroptosis and inflammation compared to males. PRs in mice lacking the BER-initiating DNA glycosylase AAG did not exhibit alkylation-induced necroptosis or inflammation. Our findings show that AAG-initiated BER at alkylated DNA bases induces sex-dependent RD primarily by triggering necroptosis and activating an inflammatory response that amplifies the original damage and, furthermore, reveal new potential targets to prevent this side effect of chemotherapy.

PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag-transgenic (AagTg) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

Parp1 protects against Aag-dependent alkylation-induced nephrotoxicity in a sex-dependent manner.

Nephrotoxicity is a common toxic side-effect of chemotherapeutic alkylating agents. Although the base excision repair (BER) pathway is essential in repairing DNA alkylation damage, under certain conditions the initiation of BER produces toxic repair intermediates that damage healthy tissues. We have shown that the alkyladenine DNA glycosylase, Aag (a.k.a. Mpg), an enzyme that initiates BER, mediates alkylation-induced whole-animal lethality and cytotoxicity in the pancreas, spleen, retina, and cerebellum, but not in the kidney. Cytotoxicity in both wild-type and Aag-transgenic mice (AagTg) was abrogated in the absence of Poly(ADP-ribose) polymerase-1 (Parp1). Here we report that Parp1-deficient mice expressing increased Aag (AagTg/Parp1-/-) develop sex-dependent kidney failure upon exposure to the alkylating agent, methyl methanesulfonate (MMS), and suffer increased whole-animal lethality compared to AagTg and wild-type mice. Macroscopic, histological, electron microscopic and immunohistochemical analyses revealed morphological kidney damage including dilated tubules, proteinaceous casts, vacuolation, collapse of the glomerular tuft, and deterioration of podocyte structure. Moreover, mice exhibited clinical signs of kidney disease indicating functional damage, including elevated blood nitrogen urea and creatinine, hypoproteinemia and proteinuria. Pharmacological Parp inhibition in AagTg mice also resulted in sensitivity to MMS-induced nephrotoxicity. These findings provide in vivo evidence that Parp1 modulates Aag-dependent MMS-induced nephrotoxicity in a sex-dependent manner and highlight the critical roles that Aag-initiated BER and Parp1 may play in determining the side-effects of chemotherapeutic alkylating agents.

Thermoregulatory correlates of nausea in rats and musk shrews.

Nausea is a prominent symptom and major cause of complaint for patients receiving anticancer chemo- or radiation therapy. The arsenal of anti-nausea drugs is limited, and their efficacy is questionable. Currently, the development of new compounds with anti-nausea activity is hampered by the lack of physiological correlates of nausea. Physiological correlates are needed because common laboratory rodents lack the vomiting reflex. Furthermore, nausea does not always lead to vomiting. Here, we report the results of studies conducted in four research centers to investigate whether nausea is associated with any specific thermoregulatory symptoms. Two species were studied: the laboratory rat, which has no vomiting reflex, and the house musk shrew (Suncus murinus), which does have a vomiting reflex. In rats, motion sickness was induced by rotating them in their individual cages in the horizontal plane (0.75 Hz, 40 min) and confirmed by reduced food consumption at the onset of dark (active) phase. In 100% of rats tested at three centers, post-rotational sickness was associated with marked (~1.5°C) hypothermia, which was associated with a short-lasting tail-skin vasodilation (skin temperature increased by ~4°C). Pretreatment with ondansetron, a serotonin 5-HT3 receptor antagonist, which is used to treat nausea in patients in chemo- or radiation therapy, attenuated hypothermia by ~30%. In shrews, motion sickness was induced by a cyclical back-and-forth motion (4 cm, 1 Hz, 15 min) and confirmed by the presence of retching and vomiting. In this model, sickness was also accompanied by marked hypothermia (~2°C). Like in rats, the hypothermic response was preceded by transient tail-skin vasodilation. In conclusion, motion sickness is accompanied by hypothermia that involves both autonomic and thermoeffector mechanisms: tail-skin vasodilation and possibly reduction of the interscapular brown adipose tissue activity. These thermoregulatory symptoms may serve as physiological correlates of nausea.