Matching target dose to target organ

A recent article by Wagner et al. reported the involvement of the anti-oxidant Nrf2 transcription factor signaling pathway in the toxicity of lead using neural stem cells in an in vitro model of neuronal differentiation¹. While this work was completed in a similar way to other studies involving in vitro lead exposure, the work avoids a critical, often neglected issue of what constitutes a relevant physiological dose in vitro. The assumption that the selected dose of 1 µM (or 20.7 ug/dL) for neuronal stem cell exposure was “4 times the CDC levels of concern (LOC) for blood lead (5 ug/dL) and is within the range of exposed populations” requires further examination. Since the in vitro exposure was completed in media (the equivalent of plasma or serum) and not in whole blood, the assumption that the in vitro lead level would be equivalent to that found in blood of lead-exposed humans is somewhat inaccurate. Lead in serum (or plasma) represents only a fraction (~1%) of the level found in whole blood^2,3, with the major fraction of lead bound inside erythrocytes⁴. For arguments sake, if the proportion of lead used in this study was 1% of that in whole blood, the equivalent blood lead value would be 2073 ug/dL, a level over 400 times the CDC LOC, and one that would be acutely toxic and perhaps lethal.

Another study, which was cited by Wagner et al.¹, showed that measurable effects in stem cells in vitro could occur at doses as low as 0.4 µM; this dose would represent a blood lead level of 800 µg/dL by our calculations⁵. In a study by Chan et al., the lowest dose of 1 µM lead used in a study of newborn rat neuronal stem cells would represent 1000 µg/L in serum and a massive systemic blood lead level of about 10,000 µg/dL⁶. Other studies examining the toxicity of lead in cell cultures have also failed to adequately match the in vitro doses^7–10 with those found in vivo, by taking account of the well documented difference between plasma and whole blood lead values. More importantly, with measurable effects on differentiation only beginning at 10 µM for Chan et al.⁶, could these data suggest the alternative interpretation that neuronal stem cells in vivo are more resistant to toxic insult by lead than our current understanding would have us believe – at least in the short term?

What is clear is that at current blood lead levels in the US population, serum or plasma levels will represent a very low fraction of those values and in vitro work could more realistically model chronic neurological effects in humans if target doses were better matched to the doses found at target sites. Thus, the model proposed in this and other work, while presenting novel effects, may be more appropriate for high acute exposures. To ensure that doses used in in vitro assays are complimentary to a target in vivo blood lead level of 20 µg/dL, exposure to cells in vitro should more accurately correspond to 1% of the blood lead value, or a dose of 0.2 µg/dL (0.01 µM). At the current CDC 5 µg/dL LOC for children, the in vitro dose would become 0.05 µg/dL (0.002 µM), a dose that would present difficulties to laboratories that cannot eliminate background levels from residual lead on glassware and other sources of possible contamination or confounding of the reported data.

In the study by Wagner et al.¹, much of this may have been considered by the authors, and key assumptions may have been made; however, the question still remains whether the upregulation of genes in the Nrf2-mediated anti-oxidative stress pathway would have been observed if a more physiologically relevant dose of 0.2 µg/dL (0.1 µM) in the media (i.e., representing a blood lead level of 20 µg/dL) had been used.

Disclaimer

The views expressed in this article are those of the author(s) and do not necessarily reflect the official policy of the Department of Defense, Department of the Army, U.S. Army Medical Department or the U.S.