## 4 Discussion
### Perpetually Adapating Cell Responses for Complementary Knowledge
#### 4.4.5 Observing Cell Behaviors  
Historically, APAP is a well-studied drug standard having known metabolites APAP-glucuron (APAP-Glu), APAP-sulfate, and APAP-glutathione (APAP-GSH) [45; 46; 47]. The targeting of APAP metabolites within the \(\blacktriangledown\)fluidic device, initially, confirmed that multiwell behaviors are consistent with known APAP compound activities as three metabolites did generate across the \(\blacktriangledown\)fluidic channel, Figures 8 and 9. Consequently, new information includes APAP concentrations versus metabolite generation levels, accruing timelines ofAPAP exposures related to up-/mid-/-downstream sites of metabolite formation, and revealing how signals from adjacent wells may influence cyclical pattens in metabolite behaviors (Figure 9). In Figure 9, APAP-GSH displays the highest cell function variability at 24 h (well 6), whereas APAP-sulfate and APAP-Glu display most function variability at 72 h in downstream culture wells 9-10.  
Previously, in 2003, Nefazodone was removed from the market after 9 years as an antidepressant product (Serzone) [78]. Discontinuation was due to adverse hepatic events and hepatic injury as listed in the World Health Organization database of adverse drug reactions, with severe reactions involving elevated bilirubin and onset of jaundice, hepatitis, and hepatocellular necrosis. With respect to time intervals, cases of hepatotoxicity usually occurred within the first 4-8 months of starting the drug. Still, the mechanism with which nefazodone causes injury remains unknown, with scientific relevance focused on P450-CYP3A4, as nefazodone is both metabolized and is an inhibitor of this enzyme. Dual functionality, induction/inhibition, indicates that competing P450-CYP3A4 drugs could delay or increase nefazodone clearance. Herein, hepatotoxicity may be mediated by toxic intermediates of its metabolism as cell byproducts influence downstream hepatic structures initiated from hepatic zone 1 (periportal), progressing through zone 2 (intermediary), and influencing zone 3 (perivenous) locals. In patient cases of acute hepatitis, liver biopsy usually demonstrated variable degrees of centroloular zone-3 necrosis [79]. By trending comparisons, the \(\upmu\)fluidic cell responses shown in Figure 7(b1-c1) display downstream cell death, an influence of cell byproducts, as upstream culture sites continue to remain viable.  
In \(\upmu\)fluidic devices, 1-way flow creates ascending compound exposures that are different from well 3 to well 11, Figure 4(b1). Gradient ramps may allow cell cultures to craft resistance against compounds. In auxiliary assessments (data not shown), select cell studies revealed two unique corollaries amid fluidic vs. static cultures: (i) cell function responses from \(\upmu\)fluidic cultures were delayed by 3-5 days prior to achieving similar outcomes as derived from static cultures, and (ii) a resistance response from \(\upmu\)fluidic cultures allowed cells to tolerate 2\(\times\)-3\(\times\) higher compound concentrations prior to harmful responses, as quantified from static cultures. Herein, the ramp of compounds may have relevance to _in vivo_ studies as the natural body ramps exposures by repeat dosing.