Polycyclic Aromatic Hydrocarbon-DNA Adducts in Gulf of Mexico Sperm Whale Skin Biopsies Collected in 2012.

The northern Gulf of Mexico has a long history of polycyclic aromatic hydrocarbon (PAH) contamination from anthropogenic activities, natural oil seepages, and the 2010 Deepwater Horizon explosion and oil spill. The continental shelf of the same area is a known breeding ground for sperm whales (Physeter macrocephalus). To evaluate PAH-DNA damage, a biomarker for potential cancer risk, we compared skin biopsies collected from Gulf of Mexico sperm whales in 2012 with skin biopsies collected from sperm whales in areas of the Pacific Ocean in 1999-2001. All samples were obtained by crossbow and comprised both epidermis and subcutaneous blubber. To evaluate exposure, 7 carcinogenic PAHs were analyzed in lipids extracted from Pacific Ocean sperm whale blubber, pooled by sex, and location. To evaluate PAH-DNA damage, portions of all tissue samples were formalin-fixed, paraffin-embedded, sectioned, and examined for PAH-DNA adducts by immunohistochemistry (IHC) using an antiserum elicited against benzo[a]pyrene-modified DNA, which crossreacts with several high molecular weight carcinogenic PAHs bound to DNA. The IHC showed widespread epidermal nuclear localization of PAH-DNA adducts in the Gulf of Mexico whales (n = 15) but not in the Pacific Ocean whales (n = 4). A standard semiquantitative scoring system revealed significantly higher PAH-DNA adducts in the Gulf of Mexico whales compared to the whales from the Pacific Ocean study (p = .0002).

In vivo localization and postmortem stability of benzo[a]pyrene-DNA adducts.

DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) play a critical role in the etiology of gastrointestinal tract cancers in humans and other species orally exposed to PAHs. Yet, the precise localization of PAH-DNA adducts in the gastrointestinal tract, and the long-term postmortem PAH-DNA adduct stability are unknown. To address these issues, the following experiment was performed. Mice were injected intraperitoneally with the PAH carcinogen benzo[a]pyrene (BP) and euthanized at 24 h. Tissues were harvested either at euthanasia (0 time), or after 4, 8, 12, 24, 48, and 168 hr (7 days) of storage at 4°C. Portions of mouse tissues were formalin-fixed, paraffin-embedded, and immunohistochemically (IHC) evaluated by incubation with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum and H-scoring. The remaining tissues were frozen, and DNA was extracted and assayed for the r7,t8,t9-trihydroxy-c-10-(N 2 -deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct using two quantitative assays, the BPDE-DNA chemiluminescence immunoassay (CIA), and high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ES-MS/MS). By IHC, which required intact nuclei, BPdG adducts were visualized in forestomach basal cells, which included gastric stem cells, for up to 7 days. In proximal small intestine villus epithelium BPdG adducts were visualized for up to 12 hr. By BPDE-DNA CIA and HPLC-ES-MS/MS, both of which used DNA for analysis and correlated well (P= 0.0001), BPdG adducts were unchanged in small intestine, forestomach, and lung stored at 4°C for up to 7 days postmortem. In addition to localization of BPdG adducts, this study reveals the feasibility of examining PAH-DNA adduct formation in wildlife species living in colder climates. Environ. Mol. Mutagen. 61:216-223, 2020. © 2019 Wiley Periodicals, Inc.

Intestinal polycyclic aromatic hydrocarbon-DNA adducts in a population of beluga whales with high levels of gastrointestinal cancers.

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) were disposed directly into the Saguenay River of the St. Lawrence Estuary (SLE) by local aluminum smelters (Quebec, Canada) for 50 years (1926-1976). PAHs in the river sediments are likely etiologically related to gastrointestinal epithelial cancers observed in 7% of 156 mature (>19-year old) adult beluga found dead along the shorelines. Because DNA adduct formation provides a critical link between exposure and cancer induction, and because PAH-DNA adducts are chemically stable, we hypothesized that SLE beluga intestine would contain PAH-DNA adducts. Using an antiserum specific for DNA modified with several carcinogenic PAHs, we stained sections of paraffin-embedded intestine from 51 SLE beluga (0-63 years), 4 Cook Inlet (CI) Alaska beluga (0-26 years), and 20 beluga (0-46 years) living in Arctic areas (Eastern Beaufort Sea, Eastern Chukchi Sea, Point Lay Alaska) and aquaria, all with low PAH contamination. Stained sections showed nuclear light-to-dark pink color indicating the presence of PAH-DNA adducts concentrated in intestinal crypt epithelial lining cells. Scoring of whole tissue sections revealed higher values for the 51 SLE beluga, compared with the 20 Arctic and aquarium beluga (P = 0.003). The H-scoring system, applied to coded individual photomicrographs, confirmed that SLE beluga and CI beluga had levels of intestinal PAH-DNA adducts significantly higher than Arctic and aquarium beluga (P = 0.003 and 0.02, respectively). Furthermore, high levels of intestinal PAH-DNA adducts in four SLE beluga with gastrointestinal cancers, considered as a group, support a link of causality between PAH exposure and intestinal cancer in SLE beluga. Environ. Mol. Mutagen. 60:29-41, 2019. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia.

Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells. Since cilia are derived from centrosomes, and aneugens can induce centrosomal amplification, the production of multiple cilia arising from multiple centrosomes may reveal the aneugenic nature of the agents. Cells were exposed to AZT to induce centrosomal amplification, cultured without serum to allow the centrioles to develop cilia, and immunostained to visualize cilia and centrosomes. Nuclear DNA was stained with DAPI. Preliminary observations suggest that cells with multiple centrosomes are able to generate extra cilia.